The  Ruling  c 

we  have  the  sqi 
nine  squares,  ea( 
leukocyte  counts 
The  very  smalles 
the  triple  ruled  1 
never  used  for  an 
in  a  vaccine.  It 
make  one  of  the 

There  are  400 
large  squares,  th 
4000  small  squar 

The  unit  in  e 
millimeter.  The 

In  making  a 
mark  1 1  just  abo 


UNIVERSITY  OF  CALIFORNIA 

MEDICAL  CENTER  LIBRARY 

SAN  FRANCISCO 


FROM  THE  IIBRARY  OF 
ARTHUR  P.  KAELBER,  M.D. 


B  first  place, 
made  up  of 
tection  with 
rge  squares. 
:ersection  of 
;are  and  are 
5  of  bacteria 
squares  to 

;re  are  nine 
There  are 

3  the  cubic 

ich  has  the 
suction  we 
solution  of 


fill  the  pipette  to , 

glacial  acetic  acid  in  water  is  most  satisfactory.     This  gives  a  dilution  of  1-20. 

Counting  with  the  2/3  inch  objective  all  of  the  highly  refractile  dots  representing 
leukocytes  in  one  of  the  i  mm.  squares  at  either  of  the  four  corners  we  note  the  num- 
ber and  mentally  multiply  by  20  (the  number  of  times  the  blood  was  diluted).  As 
the  depth  of  the  diluted  blood  between  the  ruled  surface  of  the  haemacytometer 
slide  and  the  under  surface  of  the  cover-glass  is  only  i/io  of  a  millimeter,  we  multiply 
the  figure  as  above  obtained  by  10  to  get  the  number  of  cells  in  a  1-20  dilution  of 
blood  in  a  space  of  one  cubic  millimeter. 

Example:  Counted  90  leukocytes;  90X20  =  1800X10  =  18,000:  equals  number 
of  leukocytes  in  i  cubic  mm.  of  blood. 

For  red  counts  we  use  the  red  count  pipette  which  has  the  101  mark  just  above  the 
bulb.  Taking  up  blood  to  0.5  we  draw  up  the  diluting  fluid  to  101.  This  gives  a 
dilution  of  1-200.  Counting  the  red  cells  in  five  of  the  aggregations  of  16  small 
squares  (1/20  mm.)  thus  having  counted  80  small  squares  we  have  counted  1/50 
of  the  total  number  of  small  squares  in  a  cubic  mm.,  there  being  4000  small  squares 
in  a  cubic  mm.  Consequently  the  number  of  red  cells  in  80  small  squares  multiplied 
by  50  and  then  by  the  dilution  of  200  gives  the  number  of  red  cells  in  one  cubic 
mm.  of  the  blood  examined. 

It  is  well  to  make  a  second  preparation  and  record  the  average  of  the  two  counts. 


I 


-//; 

Jtrthur  Protchf<t  Xacller  W?.  S>. 


7.5  yi 


SvwedX 


X.  ^          ^ 


•  • 


• 


Principal  normal  and  pathological  blood-cells  with  average  size, 
percentage  in  a  normal  differential  count  and  the  diseases  in  which 
certain  pathological  cells  are  more  or  less  pathognomonic. 


The  diameter  of  the  bottom  of  this  Petri  dish  is  3  inches  or  7.5+ 
centimeters. 

The  area  of  a  circle  is  equal  to  the  square  of  the  radius  multiplied 
by  TT  or  22/7. 

i  1/2  in.  =  radius,     i  1/2X1  1/2  =  2.25.     2.25X22/7  =  7.07  square 
inches. 

3.75    cm.  =  radius.     3.75X3.75  =  14-06.     14.06X22/7=44.1     square 
centimeters. 

Number  of  bacterial  colonies  in  i  sq.  in.  averages,  approximate!] 
75.     Number  in  7.07  sq.  in.  =  530. 

Number  of  bacterial  colonies  in  i  sq.  cm.  averages,  approximately, 
12.     Number  in  44.1   sq.   cm.  =  528. 

In*a  microscopic  field,  if  the  diameter  were  8  small  squares  (i/: 
mm.),  the  radius  would  be  4  small  squares  and  the  area  of  such 
round  field  would  be  4X4=16X22/7  =  50+.     Such  a  field  woi 
contain  50  small  squares. 


ire 

: 


BY  THE  SAME  AUTHOR 


The  Diagnostics  and  Treatment 

OP 

Tropical  Diseases 

86  ILLUSTRATIONS      12110     433  PAGES 
CLOTH,  $2.00,  POSTPAID 

"We  can  thoroughly  recommend  Dr.  Stitt's 
work  to  every  student  and  practitioner  of  tropical 
medicine.  It  contains  a  large  amount  of  sound 
and  up-to-date  information  presented  in  a  concise 
way  in  a  comparatively  small  space."  (From  the 
Lancet,  London.) 

"There  are  now  so  many  books  on  tropical 
medicine  that  any  fresh  addition  to  the  ranks  will 
require  to  possess  considerable  merits  to  take  a 
place  in  the  front  rank,  but  this  little  work  of 
Stitt's  will,  we  think,  attain  this  position.  Con- 
sidering the  space  at  the  author's  disposal  it  is 
wonderfully  full.  Most  of  the  subjects  are  well 
and  accurately  treated,  and  are,  in  addition,  well 
illustrated."  (From  the  British  Medical  Journal.) 

P.  BLAKISTON'S  SON  &  CO. 
PHILADELPHIA 


PRACTICAL 

Bacteriology,  Blood  Work 

AND 

Animal  Parasitology 

INCLUDING 

Bacteriological    Keys,   Zoological   Tables 
and  Explanatory  Clinical  Notes 


BY 

E.  R.  STITT,  A.  B.,  Ph.  G.,  M.  D. 

MEDICAL  DIRECTOR,  U.  S.  NAVY;  GRADUATE,  LONDON  SCHOOL  OF  TROPICAL  MEDICINE;  HEAD 
OF  DEPARTMENT  OF  TROPICAL  MEDICINE,  U.  S.  NAVAL  MEDICAL  SCHOOL;  PROFESSOR  OF 
TROPICAL  MEDICINE,  GEORGETOWN  UNIVERSITY;  PROFESSOR  OF  TROPICAL  MEDI- 
CINE, GEORGE  WASHINGTON  UNIVERSITY;  LECTURER  IN  TROPICAL  MEDICINE, 
JEFFERSON  MEDICAL  COLLEGE;  MEMBER,  NATIONAL  BOARD  OF  MEDICAL 
EXAMINERS;  MEMBER,  ADVISORY  BOARD,  HYGIENIC  LABORATORY; 
FORMERLY  ASSOCIATE  PROFESSOR    OF    MEDICAL  ZOOLOGY, 
UNIVERSITY  OF  THE  PHILIPPINES 


\   < 

Fourth  Edition,  Revised  and  Enlarged 
With  4  Plates  and  115  Other  Illustrations  Containing  505  Figures 


PHILADELPHIA 

P.   BLAKISTON'S   SON   &  CO. 

1012  WALNUT   STREET 


FIRST  EDITION,  COPYRIGHT,  1909,  BY  P.  BLAKISTON'S  SON  &  Co. 
SECOND  EDITION,  COPYRIGHT,  1910,  BY  P.  BLAKISTON'S  SON  &  Co. 

THIRD  EDITION,  COPYRIGHT,   1913,  BY  P.  BLAKISTON'S  SON  &  Co. 
FOURTH  EDITION,  COPYRIGHT,   1916,  BY  P.  BLAKISTON'S  SON  &  Co. 


TMK     M  A  I*  L  K      PKKMK     YORK     PA 


PREFACE  TO  THE  FOURTH  EDITION 

As  noted  in  previous  prefaces,  this  manual  represents  the  notes  of 
a  course  along  the  laboratory  side  of  internal  medicine  which  has 
seemed  to  the  author  practical  and  concise. 

The  exhaustion  of  a  large  edition  of  this  laboratory  guide  in  a  little 
more  than  two  years  would  indicate  a  demand  for  a  book  of  this  char- 
acter. By  combining  notes  on  points  of  clinical  importance  with 
laboratory  details  such  a  presentation  of  the  subject  would  appear  of 
more  value  to  the  student  of  internal  medicine  than  the  bare  laboratory 
technic.  . 

In  view  of  the  enormous  advances  in  internal  medicine  since  the 
appearance  of  the  last  edition  it  has  been  very  difficult  to  incorporate 
new  material  and  at  the  same  time  retain  the  pocket  manual  feature. 
This  has  been  done,  however,  by  the  greater  use  of  paragraphs  of 
smaller  type  separating  those  printed  in  larger  type,  a  plan  which  adds 
to  the  accessibility  of  the  contents  as  well  as  lessening  the  number  of 
pages.  Notwithstanding  such  utilization  of  space  it  has  been  necessary 
to  increase  the  contents  of  the  book  by  almost  one  hundred  pages. 

To  further  facilitate  quick  reference  numerous  additional  bold  type 
headings  of  paragraphs  have  been  introduced. 

It  has  been  a  difficult  matter  to  write  concise  discussions  of  such 
subjects  as  acidosis,  anaphylaxis,  deficiencies  of  renal  functioning,  etc., 
where  space  was  so  limited. 

Practically  every  chapter  in  the  book  has  been  carefully  revised 
and  new  material  added  to  each.  In  some  of  the  sections  the  changes 
in  our  views  during  the  past  two  or  three  years  have  been  so  marked 
that  it  has  been  necessary  to  entirely  rewrite  large  portions  of  such 
chapters. 

A  new  chapter,  dealing  with  diseases  of  doubtful  or  only  recently 
determined  etiology  has  been  added  to  Part  IV.  In  this  will  be  found 
discussions  of  the  vitamine  theory  in  beriberi  and  pellagra  as  well  as 
recent  findings  in  connection  with  such  diseases  as  typhus  fever,  Oroya 
fever,  verruga,  rat  bite  fever,  spotted  fever  ^pf  the  Rocky  Mountains 
and  sprue. 


fottea  lever  o 
31413 


VI  PREFACE  TO  THE  FOURTH  EDITION 

In  the  appendix  a  new  section  has  been  added  on  the  chemical  blood 
examinations,  and  in  that  on  insecticides  the  recent  views  as  to  the  best 
methods  of  destroying  lice  to  control  the  spread  of  typhus  fever  and 
relapsing  fever  have  been  incorporated. 

There  has  also  been  added  to  the  appendix  a  section  dealing  with 
anatomical  and  physiological  normals  to  furnish  ready  reference  for 
work  in  the  pathological  or  chemical  laboratory. 

Among  the  tests  more  recently  accepted  as  of  practical  value  and 
incorporated  in  this  edition  may  be  mentioned  the  following:  Schick 
test  for  diphtheria  immunity,  tests  for  recognition  of  acidosis,  tests 
for  efficiency  in  renal  functioning,  PetrofTs  method  for  culturing  tuber- 
cle bacilli,  Wolff  and  Junghans'  test  for  gastric  carcinoma,  Bronfen- 
brenner's  modification  of  Abderhalden's  technic,  tests  as  applied  to 
the  duodenal  fluid,  Lange's  colloidal  gold  test  for  general  paresis, 
Fontana's  spirochete  staining  technic,  Gluzinski's  gastric  carcinoma 
test,  and  many  others. 

There  have  also  been  many  new  illustrations  of  animal  parasites 
substituted  for  those  in  the  third  edition  which  did  not  appear  to  have 
sufficient  teaching  value. 

E.  R.  S. 


PREFACE  TO  THE  THIRD  EDITION 

IN  the  preparation  of  the  third  edition  of  this  laboratory  manual  it 
soon  became  evident  that  the  new  material  to  be  added  would  increase 
the  size  of  the  book  beyond  that  which  would  permit  its  being  readily 
carried  in  one's  pocket.  It  has,  however,  been  possible  to  keep  the 
size  of  the  book  within  the  limits  considered  desirable  by  the  use  of  a 
smaller  type  in  a  considerable  proportion  of  the  paragraphs  so  that  in 
this  way  and  by  increasing  the  number  of  lines  on  each  page  it  has  been 
possible  to  add  extensively  to  the  subject  matter  and  with  only  an  in- 
crease of  about  sixty-five  pages. 

The  advantage  attaching  to  more  ready  reference  obtained  by  the 
alternation  of  different  sizes  of  type  would  appear  to  make  this  plan  an 
improvement  over  the  old. 

While  the  chapters  dealing  with  bacteriology  have  been  added  to 
and  made  to  include  more  recent  advances  it  will  be  noted  that  in  the 
section  on  animal  parasitology  the  subject  matter  has  been  greatly 
increased. 

In  the  revision  of  the  chapter  on  protozoa  I  am  greatly  indebted  to 
Professor  Minchin's  recent  work  on  the  Protozoa  and  in  those  relating 
to  arachnoids  and  insects  to  the  very  practical  volume  of  Colonel 
Alcock  entitled  "Entomology  for  Medical  Officers." 

The  illustrations  have"  been  added  to  and  many  which  did  not  seem 
to  bring  out  sufficiently  details  of  anatomy  have  been  replaced  by  others 
more  satisfactory  in  that  respect. 

The  three  plates  of  the  cestode,  trematode  and  nematode  ova  were 
drawn  by  Mr.  L.  Avery  under  the  supervision  of  P.  A.  Surgeon  Garrison, 
U.  S.  N.  and  it  is  believed  that  they  will  be  found  more  satisfactory 
than  similar  plates  contained  in  works  on  animal  parasitology. 

Several  new  tables  have  been  added  among  which  special  attention 
is  called  to  the  one  on  urinary  findings  in  various  diseases  of  the  genito- 
urinary system  and  also  to  the  key  to  the  intestinal  bacteria  attached 
to  the  inside  of  the  board  cover. 

A  chapter  on  "Disinfectants  and  Insecticides"  giving  the  practical 
application  of  methods  of  carrying  out  these  important  Public  Health 
questions  has  been  added. 

vii 


Vlii  PREFACE  TO  THE  THIRD  EDITION 

In  the  chapter  on  "Immunity"  a  modification  of  Emery's  technic 
for  the  Wassermann  test  has  been  incorporated — the  use  of  Noguchi's 
reagents  with  Emery's  technic.  The  subject  matter  of  the  sections  on 
vaccines  and  anaphylaxis  has  been  extensively  revised. 

E.  R.  S. 


PREFACE  TO  THE  SECOND  EDITION 

THE  fact  of  the  necessity  for  a  second  edition  of  this  manual  of  lab- 
oratory and  clinical  diagnosis  in  a  little  more  than  a  year  would  indicate 
that  the  original  arrangement  of  material  should  be  adhered  to. 

Each  section  of  the  book  had  been  carefully  revised  and  much  new 
matter  added.  In  particular  has  that  part  of  the  book  relating  to  ani- 
mal parasitology  been  rewritten  and  almost  doubled  in  extent,  and  a 
chapter  on  Poisonous  Snakes  added.  In  the  chapter  on  "  Practical 
Methods  in  Immunity"  the  most  recent  advances  in  the  Wassermann 
test  and  practical  agglutination  methods  have  been  incorporated  as  well 
as  a  brief  discussion  of  the  question  of  Anaphylaxis. 

The  section  on  "  Clinical  Bacteriology  and  Animal  Parasitology  of 
the  Various  Body  Fluids  and  Organs"  has  been  revised  to  meet  the  most 
recent  advances  in  clinical  diagnosis.  This  section  not  only  answers 
as  a  cross  index  to  the  importance  of  the  various  bacteria  and  animal 
parasites  in  practical  clinical  work,  but  gives  a  concise,  practical  state- 
ment as  to  how  to  proceed  in  the  examination  of  various  secretions  and 
excretions.  This  information  is  difficult  to  obtain  in  the  larger  works 
on  clinical  diagnosis  by  reason  of  its  being  taken  up  under  many  differ- 
ent headings. 

A  method  is  given  for  the  making  of  differential  counts  in  the  same 
preparation  as  that  for  making  the  leukocyte  count  which  has  the  ad- 
vantages of  accuracy  and  the  saving  of  time. 

Several  new  illustrations  have  been  added — the  one  of  poisonous 
snakes  has  been  taken  from  Stejneger's  report. 

The  plan  of  making  this  little  volume  a  practical  one  has  been  con- 
tinued in  the  second  edition;  theoretical  considerations  have  been 
brought  out  only  when  necessary  to  a  proper  understanding  of  some 
recent  or  difficult  laboratory  method. 

The  very  elementary  considerations  and  definitions  have  not  been 
given  because  in  order  to  present  a  compact  and  at  the  same  time  a 
practical  working  guide  it  has  been  necessary  to  eliminate  that  which 
seemed  least  essential.  Furthermore,  instruction  in  biological  science 
is  now  a  part  of  the  requirements  of  candidates  for  admission  to  the 
various  medical  schools. 

ix 


X  PREFACE  TO  THE  SECOND  EDITION 

At  the  request  of  many  who  have  found  the  book  of  assistance  I 
have  added  an  outline  of  those  methods  in  the  chemical  examination 
of  urine  and  gastric  contents  which  have  seemed  to  me  to  be  most  essen- 
tial in  the  making  of  diagnoses.  In  the  tropics  I  have  found  the  deter- 
minations of  total  nitrogen  and  nitrogen  eliminated  as  ammonia  to  be 
exceedingly  valuable  in  diagnosis.  Methods  for  such  determinations, 
as  elaborated  by  Assistant  Surgeon  E.  W.  Brown,  U.  S.  Navy,  of  the 
U.  S.  Naval  Medical  School,  have  proven  satisfactory  and  have  been 
incorporated  in  this  section  which  is  to  be  found  in  the  Appendix. 

Every  effort  has  been  made  to  keep  the  book  within  the  limits  of  a 
pocket  manual. 

Owing  to  my  absence  from  the  United  States  I  have  to  thank  Dr. 
Charles  S.  Butler  for  correcting  the  proof.  For  the  revision  of  the 
index  I  am  indebted  to  Mr.  John  P.  Griest. 

E.  R.  S. 


PREFACE  TO  THE  FIRST  EDITION 

WHILE  a  member  of  the  Naval  Examining  Board  and  examiner  in 
bacteriology  and  clinical  microscopy,  I  have  during  the  past  six  years 
had  an  opportunity  to  judge  of  the  qualifications  of  several  hundred 
graduates  of  the  various  medical  schools  of  the  country  from  the  stand- 
point of  practical  application  in  the  laboratory  of  that  which  they  had 
learned  as  undergraduates. 

More  particularly  I  have  made  it  a  point  to  ascertain  from  the  suc- 
cessful candidates,  while  under  instruction  at  the  Naval  Medical  School, 
the  features  of  their  laboratory  courses,  which  had  seemed  to  them 
most  practical;  such  methods  being  subsequently  tested  in  our  own 
class  work. 

As  a  result  I  have  endeavored  to  incorporate  in  this  manual  methods 
which  have  been  submitted  to  the  criticism  of  postgraduate  students 
from  all  the  leading  medical  schools  of  the  country,  and  which  have  been 
considered  by  them  adapted  to  the  requirements  of  practical,  speedy, 
and  satisfactory  clinical  laboratory  diagnosis. 

For  the  laboratory  worker  the  most  valuable  asset  is  common  sense 
and  he  must  be  able  to  bring  to  mind  the  possibilities  of  the  production 
of  various  artefacts  and  results  from  trivial  errors  in  technic.  It  has 
been  my  object  to  point  out  where  such  mistakes  may  arise,  the  reasons 
for  obtaining  results  differing  from  those  ordinarily  obtained  and  the 
means  employed  to  eliminate  as  far  as  possible  such  results. 

We  are  too  apt  to  neglect  the  trivial  details  of  stains,  reaction  of 
media,  and  the  like,  yet  it  is  only  when  every  detail  of  technic  has  been 
rigidly  carried  out  that  we  are  in  a  position  to  judge  of  the  significance 
of  an  object  observed  in  a  microscopical  preparation. 

In  bacteriology,  candidates  were  frequently  able  to  give  the  cul- 
tural and  morphological  characteristics  of  all  the  important  pathogenic 
organisms,  yet  when  it  was  required  of  them  to  outline  the  procedure 
by  which  they  would  differentiate  members  of  the  typhoid-colon  groups 
when  encountered  in  a  plate  made  from  faeces,  the  problem  appeared 
to  them  impossible.  They  possessed  the  information,  but  did  not  know 
how  to  apply  it. 

In  practical  work,  organisms  can  only  be  separated  culturally  by  the 
use  of  Keys  and  for  this  reason  Keys  are  given  at  the  beginning  of  each 

xi 


xii  PREFACE  TO  THE  FIRST  EDITION 

division  of  bacteria.  These  enable  one  to  quickly  place  the  organism 
isolated  in  its  respective  group.  Only  methods  of  differentiation  which 
are  applicable  in  a  physician's  private  laboratory  are  given.  Practical 
methods  for  making  the  final  identification  by  agglutination  or  other 
immunity  tests  are  described.  Technic  for  immunizing  animals  to 
furnish  such  sera  is  given  in  detail. 

The  giving  of  the  cultural  characteristics  in  a  systematic  tabulated 
Key  gives  space  in  the  notes  for  presenting  the  salient  points  in  the 
pathological  and  epidemiological  aspects  of  each  organism. 

I  have  endeavored  to  give  a  scientific  yet  practical  classification  of 
the  important  pathogenic  moulds,  a  subject  about  which  there  exists 
greater  confusion  in  the  minds  of  students  than  for  any  other.  In  the 
nomenclature  I  have  followed  Gedoelst's  "Les  Champignons  Parasites." 

In  the  chapter  on  Media  Making,  it  is  believed  that  anyone  after 
reading  this  section  and  following  the  instructions  will  be  able  to  satis- 
factorily and  without  the  adjuncts  of  a  large  laboratory  make  any  kind 
of  media.  The  directions  as  to  titrations  are  given  in  detail  because 
it  is  beginning  to  be  recognized  that  reaction  of  media  in  bacteriology 
is  of  as  great  importance  as  staining  is  in  blood  work. 

The  section  on  Blood  Work  is  practical  and  gives  a  method  for  mak- 
ing a  Romanowsky  stain  which  is  quick  and  reliable.  The  chapter  on 
Normal  and  Pathological  Blood  gives  in  a  few  pages  the  more  important 
points  to  be  borne  in  mind  in  considering  a  possible  diagnosis. 

While  there  is  no  difference  between  the  laboratory  requirements  of 
medical  work  in  the  tropics  and  that  in  temperate  climates,  unless  by 
reason  of  such  measures  of  diagnosis  being  indispensable  in  the  tropics, 
it  has,  however,  been  my  endeavor  to  treat  every  tropical  question, 
whether  in  blood  work,  bacteriology,  or  animal  parasitology,  in  a  more 
complete  way  than  is  usual  in  manuals  of  this  character.  Therefore  it 
is  believed  that  this  little  book  will  be  of  great  service  to  the  laboratory 
worker  in  the  tropics. 

It  is  only  from  working  under  Doctor  Charles  W.  Stiles  in  his  course 
of  laboratory  instruction  in  Animal  Parasitology  in  the  United  States 
Naval  Medical  School  that  I  feel  justified  in  presenting  a  concise  out- 
line of  the  subjects  in  medical  zoology  which  appear  to  me  to  be  most 
important  for  the  physician. 

The  system  of  arranging  tables,  showing  the  families,  genera,  etc., 
in  which  each  species  belongs  will,  it  is  believed,  greatly  simplify  the 
matter  of  classification  for  the  medical  student.  The  points  given 


PREFACE  TO  THE  FIRST  EDITION  xiii 

under  each  parasite  are  believed  to  be  practical  ones.  When  a  parasite 
has  only  been  reported  for  man  two  or  three  times,  very  little  space  is 
given  to  it. 

Part  IV  summarizes  the  various  infections  which  may  be  found  in 
different  organs  or  excretions  of  the  body  and  embraces  both  bacterial 
and  animal  parasites.  Practical  methods  for  examining  material  are 
also  given. 

The  chapter  on  Immunity,  in  which  the  theoretical  side  is  immedi- 
ately illustrated  by  the  practical  application  will  tend  to  simplify  this 
bug-bear  of  the  medical  student. 

The  illustrations  have  been  selected  with  a  view  to  bringing  out 
points  which  are  difficult  to  state  briefly  in  the  text,  and  furthermore 
they  have  been  grouped  together  so  that  comparison  of  similar  parasites 
is  possible  without  turning  from  page  to  page. 

I  have  in  particular  to  thank  Hospital  Steward  Ebeling  of  the  Navy 
for  his  care  in  bringing  out  such  details. 

By  reason  of  the  authority  of  Braun,  it  has  been  considered  sufficient 
to  give  in  the  tables  only  the  proper  zoological  name  of  the  parasite  as 
given  in  the  1908  German  edition.  The  synonyms  have  been  omitted 
for  consideration  of  space. 

The  works  chiefly  consulted  in  addition  to  that  of  Braun  have  been : 
Albutt's  System  of  Medicine;  Osier's  System  of  Medicine;  Muir  and 
Ritchie's  Bacteriology;  Mense's  Tropenkrankheiten;  Blanchard's  Les 
Moustiques;  Guiart  and  Grimbert's  Diagnostic;  Ehrlich's  Studies  in 
Immunity;  Stephens  and  Christopher's  Practical  Study  of  Malaria; 
Daniel's  Laboratory  Studies  in  Tropical  Medicine;  Manson's  Tropical 
Diseases;  Gedoelst's  Les  Champignons  Parasites;  Neveu-Lemaire 
Parasitologie  Humaine;  Chester's  Determinative  Bacteriology;  Leh- 
mann  and  Neumann's  Bacteriology. 

E.  R.  S. 


CONTENTS 

PART  I.     BACTERIOLOGY 

CHAPTER  I. — APPARATUS. 

The  Microscope,  i — Apparatus  for  sterilization,  6 — Cleaning  glass- 
ware, 9 — Concave  slides,  fermentation  tubes,  n. 

CHAPTER  II. — CULTURE  MEDIA. 

Nutrient  bouillon,  20 — Standardization  of  reaction,  21 — Sugar-free 
bouillon,  23 — Glycerine  bouillon,  25 — Peptone  solution,  25 — Nutrient 
agar,  26 — Glycerine  agar  egg  medium,  27 — Gelatine,  28 — Litmus  milk, 
28 — Potato,  29 — Blood  serum,  30 — Blood  agar,  31 — Bile  and  faeces 
media,  31-33 — Culture  media  for  protozoa,  35. 

CHAPTER  III. — STAINING  METHODS. 

Loffler's  methylene  blue,  39 — Carbol  fuchsin,  39 — Gram's  method,  39 
— Acid-fast  staining,  41 — Neisser's  stain,  42 — Capsule  staining,  43 — 
Flagella  staining,  44 — Spore  staining,  45 — Staining  of  protozoa,  45. 

CHAPTER  IV. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.    GENERAL  CON- 
SIDERATIONS. 
Methods  of  isolating  bacteria,  48 — Classification,  50 — Use  of  keys,  51. 

CHAPTER  V. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.     Cocci. 

Key,  57 — Streptococci,  58 — Sarcinae,  62 — Staphylococci,  63 — Pneu- 
mococcus,  64 — Gram-negative  cocci,  66. 

CHAPTER  VI. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.     SPORE-BEARING 

BACILLI. 

Key,  73 — Anthrax,  75 — Cultivation  of  anaerobes,  78 — Malignant 
cedema,  82 — B.  botulinus,  82 — B.  tetani,  84 — B.  aerogenes  capsulatus, 

87- 

CHAPTER  VII. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.     BRANCHING, 
CURVING  BACILLI.     MYCOBACTERIA.     CORNYEBACTERIA. 

Acid-fast  bacilli,  91 — Tubercle  bacillus,  92 — Leprosy  bacillus,  97 — 

Non-acid-fast  branching  bacilli,  101 — B.  mallei,  101 — B.  diphtherias, 

102 — Hofmann's  bacillus,  107 — B.  xerosis,  108. 
CHAPTER  VIII. — STUDY  AND  IDENTIFICATION  OF  BACTERIA. 

Gram-negative  bacilli,  Hemophilic  bacteria,  in — Influenza  bacillus, 

in — Priedlander's  bacillus,  114 — Plague,  114 — Eberth,  Gartner,  and 

Escherich  groups,  118 — Typhoid,  119 — Dysentery,  125 — Chromogenic 

bacilli,  129. 
CHAPTER  IX. — STUDY  AND  IDENTIFICATION  OF  BACTERIA. 

Spirilla,  131 — Cholera,  131. 

CHAPTER  X. — STUDY  AND  IDENTIFICATION  OF  MOULDS,  136. 
CHAPTER  XI. — BACTERIOLOGY  OF  WATER,  AIR,  AND  MILK. 

Water,  149 — Milk,  155 — Air,  159. 

xv 


Xvi  CONTENTS 

CHAPTER  XII. — PRACTICAL  METHODS  OF  IMMUNITY. 

Methods  of  obtaining  immune  sera,  165 — Agglutination  tests,  168 — 
Precipitins,  170 — Deviation  of  the  Complement,  171 — Fixation  of 
the  Complement,  172 — The  Wassermann  reaction,  174 — Opsonic 
power  and  preparation  of  vaccines,  186 — Anaphylaxis,  192. 

PART  II.     STUDY  OF  THE  BLOOD 

CHAPTER    XIII. — MlCROMETRY   AND    BLOOD    PREPARATIONS. 

Micrometry,  197 — Haemoglobin  estimation,  200 — Counting  blood,  202 
— Study  of  fresh  blood,  206 — Blood  films,  208 — Staining  blood  films, 
211 — lodophilia,  214 — Occult  blood,  216 — Acidosis,  218. 
CHAPTER  XIV. — NORMAL  AND  PATHOLOGICAL  BLOOD. 

Color  index,  222 — Red  cells,  222 — White  cells,  224 — Eosinophilia,  231 
— Leukocytosis,  232 — Lymphocytosis,  234 — Diseases  with  a  normal 
leukocyte  count,  235 — The  primary  anaemias,  235 — Secondary  anae- 
mias, 237 — The  leukemias,  238. 

PART  III.    ANIMAL  PARASITOLOGY 

CHAPTER  XV. — CLASSIFICATION  AND  METHODS,  243. 

CHAPTER  XVI.— THE  PROTOZOA. 

Rhizopoda,  251 — Flagellata,  259 — Infusoria,  273 — Sporozoa,  282— 
The  malarial  parasite,  283. 

CHAPTER  XVII.— THE  FLAT  WORMS. 

Flukes,  294 — Liver  flukes,  297 — Intestinal  flukes,  298 — Lung  flukes, 
299 — Blood  flukes,  300 — Cestodes,  303 — Somatic  taeniasis,  310. 

CHAPTER  XVIII.— THE  ROUND  WORMS. 

Filariidae,  315 — Key  to  filarial  larvae,  319 — Trichinosis,  321 — Hook- 
worms, 325 — Ascaridae,  330 — Leeches,  332. 

CHAPTER  XIX. — THE  ARACHNOIDEA. 

The  mites,  335 — The  ticks,  338 — The  Linguatulidae,  342. 

CHAPTER  XX.— THE  INSECTS. 

The  Pediculidae,  345— The  Diptera,  351— Biting  flies,  352— Myiases, 

359- 
CHAPTER  XXI.— THE  MOSQUITOES. 

Dissection  of  mosquitoes,  370 — Differentiation  of  Culicinae  and  Anoph- 

elinae,  371 — Classification  of  Culicidae,  372. 
CHAPTER  XXII.— THE  POISONOUS  SNAKES,  377. 

PART  IV.     CLINICAL  BACTERIOLOGY  AND  ANIMAL 

PARASITOLOGY  OF  THE  VARIOUS  BODY 

FLUIDS  AND  ORGANS 

CHAPTER  XXIII.— DIAGNOSIS  OF  INFECTIONS  OF  THE  OCULAR  REGION,  381. 
CHAPTER  XXIV. — DIAGNOSIS  OF  INFECTIONS  OF  THE  NASAL  CAVITIES,  384. 
CHAPTER  XXV. — EXAMINATION  OF  BUCCAL  AND  PHARYNGEAL  MATERIAL, 

386. 
CHAPTER  XXVI.— EXAMINATION  OF  SPUTUM,  389. 


CONTENTS  XV11 

CHAPTER  XXVII. — THE  URINE,  393 — RENAL  FUNCTIONING,  402. 

CHAPTER  XXVIII.— THE  FAECES,  405. 

CHAPTER  XXIX.— BLOOD  CULTURES  AND  BLOOD  PARASITES,  412. 

CHAPTER  XXX. — THE  STOMACH  CONTENTS,  416 — DUODENAL  FLUID,  417. 

CHAPTER  XXXI. — EXAMINATION  OF  Pus,  419. 

CHAPTER  XXXII. — SKIN  INFECTIONS,  421. 

CHAPTER  XXXIII.— CYTODIAGNOSIS,  423— SPINAL  FLUID,  425. 

CHAPTER  XXXIV. — RABIES,  VACCINIA  AND  SMALLPOX,  429 — FILTERABLE 

VIRUSES,  433. 
CHAPTER  XXXV.— DISEASES  OF  UNKNOWN  ORIGIN,  435. 

APPENDIX 

PREPARATION  OF  TISSUES  FOR  EXAMINATION  IN  MICROSCOPIC  SECTIONS,  443. 

MOUNTING  AND  PRESERVATION  OF  ANIMAL  PARASITES,  450. 

PREPARATION  OF  NORMAL  SOLUTIONS,  452. 

CHEMICAL  EXAMINATION  OF  BLOOD,  454. 

CHEMICAL  EXAMINATION  OF  THE  URINE,  456. 

CHEMICAL  EXAMINATION  OF  THE  GASTRIC  CONTENTS,  469. 

CHEMICAL  TESTS  OF  DUODENAL  FLUID,  472. 

DISINFECTANTS  AND  INSECTICIDES,  472. 


BACTERIOLOGY,  BLOOD-WORK   AND 
ANIMAL  PARASITOLOGY 


CHAPTER  I 
APPAJRATUS 

THE  MICROSCOPE 

THE  most  important  piece  of  apparatus  for  the  laboratory  worker 
is  the  microscope.  Very  satisfactory  microscopes  can  be  purchased  in 
this  country.  Instruments  of  standard  German  make  are  in  use  in 
many  laboratories  and  appear  to  give  general  satisfaction.  It  is  impos- 
sible to  do  good  microscopical  work  unless  the  microscope  gives  and 
continues  to  give  good  definition  and  the  working  parts  remain  firm. 

Folding  microscope  stands  are  now  made  which  are  perfectly  satisfactory,  such 
instruments,  however,  have  only  the  advantage  of  occupying  less  space  in  a  case  so 
that  unless  the  question  of  compactness  is  involved,  as  in  an  outfit  for  the  military 
services  or  for  a  microscopist  who  travels  about  a  great  deal,  the  ordinary  rigid  horse- 
shoe base  is  to  be  preferred. 

A  mechanical  stage  is  almost  a  necessity  in  connection  with  blood-work  and  its  use 
is  advantageous  in  bacterial  preparations.  For  the  study  of  tissue  sections  the  mov- 
ing of  the  slide  with  the  fingers  is  preferable.  Therefore,  the  mechanical  stage  should 
be  capable  of  ready  attachment  or  removal.  For  the  examination  of  colonies 
growing  in  Petri  dishes  we  also  use  the  stage  unencumbered  with  the  mechanical 
stage.  A  triple  or  quadruple  nose-piece,  according  to  the  number  of  objectives  used, 
is  also  indispensable. 

One  should  always  use  a  magnifying  glass  or  better  a  lens  in  a  tripod  or  an  apla- 
natic^triplet  in  the  study  of  microscopic  objects,  prior  to  examination  with  the  mi- 
croscope. A  dissecting  microscope  is  better  for  this  purpose  and  is  very  useful  in 
dissecting  mosquitoes,  etc.  In  fact  the  dissecting  microscope  is  almost  essential  in 
the  examination  of  helminthological  and  entomological  specimens.  Of  particular 
value  is  a  stage  forceps  for  orienting  insects,  especially  mosquitoes,  when  examined 
on  the  stage  of  either  a  compound  or  simple  microscope. 

The  following  precautions  should  be  observed  to  prevent  injury  to 
the  microscope: 

i.  If  the  fine  adjustment  works  through  the  arm  of  the  microscope,  always  grasp 
the  instrument  by  the  pillar  which  supports  the  stage.  In  those  microscopes,  how- 


2  APPARATUS 

ever,  which  are  not  constructed  in  this  way  the  arm  is  made  to  serve  in  lifting  the 
instrument. 

2.  Always  keep  the  microscope  in  its  case  or  covered  with  a  bell  jar  when  not  in 
use  in  order  to  keep  away  the  dust.     A  piece  of  black  cloth  supported  on  a  wire 
lamp-shade  frame  makes  a  most  convenient  protecting  covering. 

3.  Alcohol  ruins  the  lacquer  of  an  instrument  and  care  should  be  observed  to  keep 
all  parts  of  the  microscope  from  coming  in  contact  with  acids,  alkalies,  chloroform  or 
xylol  as  well  as  alcohol. 

4.  Always  use  Japanese  lens  paper  in  wiping  off  the  dust  from  dry  objectives  or 
the  immersion  oil  from  the  2-mm.  one.     Should  one  neglect  to  wipe  off  the  oil  from 
the  oil-immersion  objective,  the  dried  oil  can  be  removed  by  wiping  with  a  drop  of 
xylol  on  lens  paper,  but  the  cleaning  should  be  done  as  rapidly  as  possible,  with  a 
final  wiping  off  with  dry  lens  paper,  to  avoid  damage  by  the  xylol  to  the  setting  of 
the  lenses.     Throw  lens  paper  away  after  using  it  once. 

5.  Lenses  are  very  liable  to  deteriorate  in  the  tropics.     One  should  be  careful  to 
protect  his  instrument  from  the  direct  light  of  the  tropical  sun. 

6.  If  any  oil  is  used  on  the  mechanical  parts  for  lubrication,  all  excess  should  be 
wiped  off  to  avoid  the  catching  of  dust  or  gritty  particles. 

Objectives. — To  meet  the  demands  of  clinical  microscopy  there 
should  be  three  objectives,  preferably  a  i6-mm.  (%-inch),  a  4-mm. 
(%-inch)  and  a  2-mm.  (J^-indi)  homogeneous  oil  immersion  one. 

The  Zeiss  A  A  is  a  ly-mm.  objective,  and  the  Leitz  No.  3,  an  i8-mm.  or  ^-inch 
one.  The  Zeiss  D  is  about  4. 2-mm.  and  the  Leitz  No.  6,  4.4-mm.  or  ^-inch.  A 
dustproof  quadruple  nose-piece  with  four  objectives  will  be  found  a  great  convenience 
(in  addition  to  the  %-inch  and  ^2-mcn  objectives,  a  ^-inch  for  urine  and  blood 
counting,  with  a  ^-inch  for  examining  hanging-drop  preparations  and  for  quick 
examination  of  blood  smears).  An  apochromatic  objective  costs  about  three  times 
as  much  as  an  achromatic  one  and,  except  in  photographic  work,  has  little  if  any 
advantage. 

Oculars. — As  regards  oculars  (eye-pieces)  a  Leitz  No.  i  and  a  No.  4 
will  best  meet  the  requirements.  For  high  magnification  a  No.  8  may 
be  of  service.  The  Zeiss  oculars  are  numbered  according  to  the  amount 
they  increase  the  magnification  given  by  the  objective;  thus  a  No.  2 
increases  the  magnification,  given  by  the  objective  alone,  twice;  a  No. 
8,  eight  times. 

Some  oculars  are  classified  according  to  their  equivalent  focal  distance  and  are 
referred  to  as  H-inch,  i-inch  and  2 -inch  oculars. 

A  i-inch  or  25-mm.  ocular  magnifies  the  magnification  produced  by  the  objective 
about  10  times  while  a  2-inch  or  5o-mm.  one  increases  the  magnification  of  the  ob- 
jective four  times. 

A  Leitz  No.  o  is  a  5o-mm.  ocular  and  magnifies  four  times.  The  Nos.  i,  2,  3,  4 
and  5  are  40,  35,  25,  and  20  mm.  respectively  and  give  eye-piece  magnifications  of  5, 
6,  8, 10  and  12  times. 

The  oculars  in  common  use  are  known  as  negative  or  Huyghenian  oculars,  by 


OBJECTIVES  3 

which  is  meant  an  ocular  in  which  the  lower  lens  (collective)  assists  in  forming  the  real 
inverted  image  which  is  focused  at  the  level  of  the  diaphragm  within  the  ocular. 
When  using  a  disc  micrometer,  it  is  supported  by  this  diaphragm,  and  the  outlines  of 
the  image  are  cut  by  the  rulings  on  the  glass  disc,  and  so  we  are  enabled  to  measure 
the  size  of  the  object  examined.  The  measurement  of  various  bacteria,  blood-cells, 
and  parasites  is  exceedingly  simple  and  assists  greatly  in  the  study  of  bacteria,  and  is 
indispensable  in  work  in  animal  parasitology.  (For  details  of  micrometry  see  section 
on  blood- work.)  When  an  ocular  is  termed  positive,  it  refers  to  an  ocular  which  acts 
as  a  simple  microscope  in  magnifying  the  image,  the  image  being  formed  entirely  by 
the  objective  and  located  below  the  ocular.  By  fixing  one  end  of  a  hair  on  the  rim  of 
the  diaphragm  inside  the  ocular  with  a  minute  drop  of  balsam  one  has  a  satisfactory 
pointer  to  locate  any  particular  cell  in  the  microscopic  field. 

Objectives  are  usually  designated  by  their  equivalent  focal  distance.  It  is  impor- 
tant to  remember  that  the  equivalent  focal  distance  does  not  represent  the  working 
distance  of  an  objective,  by  which  is  meant  the  distance  from  the  upper  surface  of 
the  cover-glass  to  the  lower  surface  of  the  objective.  Thus  a  K-inch  objective  may 
have  to  be  approached  to  the  object  so  that  the  distance  intervening  may  be  only 
%  inch  or  even  less.  This  explains  the  frequent  inability  to  focus  an  object  when  a 
high-power  dry  objective  (^-inch  or  ^-inch)  is  used  with  a  rather  thick  cover-glass 
— the  objective  possibly  having  a  short  working  distance,  so  that  the  thickness  of 
the  cover-glass  does  not  allow  of  any  free  working  distance. 

Instrument  makers  generally  specify  the  thickness  of  cover-glass  to  be  used  with 
a  certain  tube  length,  but  as  a  practical  matter  it  will  be  found  convenient  to  use  No . 
i  (very  thin)  cover-glasses.  The  principal  objection  to  these  is  that  they  are  more 
fragile  than  the  No.  2,  but  with  a  little  practice  in  cleaning  cover-glasses  this  is 
negligible.  Immersion  lenses  are  less  affected  than  dry  lenses  by  the  question  of  a 
certain  thickness  of  cover-glasses  for  a  certain  tube  length. 

One  of  the  most  fruitful  causes  of  the  crushing  of  microscopical  ob- 
jects and  the  overlying  cover-glass  or,  what  is  far  more  important,  the 
breaking  of  the  cover-glass  of  a  hanging-drop  preparation  and  conse- 
quent risk  of  infection  is  the  attempt  to  focus  with  the  fine  adjustment. 

It  should  be  an  invariable  rule  for  the  worker  to  bring  his  objective  practically  into 
contact  with  the  upper  surface  of  the  cover-glass,  then  using  the  coarse  adjustment 
(rack  and  pinion)  to  slowly  elevate  the  objective,  looking  through  the  eye-piece  at 
the  same  time.  In  other  words,  obtain  focus  with  the  coarse  adjustment  and  main- 
tain it  with  the  fine  adjustment  (micrometer  screw).  The  fine  adjustment  should 
only  be  used  after  the  focus  is  obtained. 

Oil  Immersion. — In  using  the  oil-immersion  objective  always  dip  the 
lens  in  the  oil  and  practically  touch  the  cover-glass — the  eye  being  at 
a  level  with  the  stage — before  beginning  to  focus.  With  the  coarse  ad- 
justment one  can  feel  the  contact  with  the  cover-glass,  which  is  impos- 
sible with  the  fine  adjustment.  It  saves  time  and  disappointment  to 
make  a  preliminary  examination  of  a  preparation  requiring  the  high 


4  APPARATUS 

dry  or  immersion  lens  with  a  low  power  (%-inch)  before  employing  the 
higher  power;  in  this  way  we  locate  or  center  a  suitable  field  for  study. 

It  will  be  observed  that  objectives  frequently  have  their  numerical  aperture 
marked  on  them.  This  is  expressed  by  the  letters  N.A.  From  a  practical  stand- 
point this  gives  the  relative  proportion  of  the  rays  which  proceeding  from  an  object 
can  enter  the  lens  of  the  objective  and  form  the  image.  Of  course,  the  greater  the 
number  of  rays,  the  greater  the  N.A.,  the  better  the  definition,  and  consequently  the 
better  the  objective.  Immersion  oil,  having  the  same  index  of  refraction  (1.52) 
as  glass,  would  not  deflect  rays  coming  from  the  object  and  so  prevent  their  enter- 
ing the  objective,  as  would  be  the  case  if  we  used  a  dry  objective  with  an  interven- 
ing air  space.  In  this  case  a  portion  of  the  rays  would  be  turned  aside  by  the  differ- 
ence in  the  refractive  index  of  air.  As  a  rule,  the  higher  the  numerical  aperture, 
the  better  the  objective  and  the  less  the  working  distance.  In  blood  counting,  the 
cover-glass  being  comparatively  thick,  it  may  happen  that  with  a  ^j-inch  of  high 
numerical  aperture  there  may  not  be  sufficient  working  distance  to  bring  the  blood- 
cells  into  focus,  which  could  be  done  with  an  objective  of  lower  numerical  aperture. 
Consequently,  we  must  always  consider  the  matter  of  working  distance  as  well  as 
that  of  numerical  aperture.  The  skill  of  the  optician,  however,  can  obviate  this 
defect  in  an  objective  of  high  numerical  aperture  so  that  it  may  combine  the  qualities 
of  perfect  definition  with  sufficient  working  distance. 

Practical  Points  in  the  Use  of  the  Microscope. — An  important  matter 
in  the  use  of  the  microscope  is  to  get  all  the  details  possible  with  a  low 
power  before  using  a  higher  power.  This,  of  course,  does  not  apply  to  a 
bacterial  preparation  where  it  is  necessary  to  use  a  J^2~mch  or  a  high- 
power  dry  lens. 

It  is  well,  however,  in  a  bacterial  or  blood  preparation  to  first  examine  the  smear 
with  the  %-inch  objective  in  order  to  determine  suitable  areas  for  examination  with 
the  oil-immersion  objective.  With  tissue  sections  it  is  not  only  advisable  to  begin  the 
study  with  the  lowest  power,  but  even  an  examination  with  the  unaided  eye  or  with 
a  magnifying  glass,  before  using  the  microscope,  will  give  a  surprising  amount  of 
information. 

After  using  the  oil-immersion  objective  the  lens  should  be  wiped  clean  of  oil 
with  a  strip  of  Japanese  lens  paper  or  with  a  silk  handkerchief.  If  the  oil  should 
dry  on  the  surface  of  the  lens  it  may  be  removed  with  a  drop  of  xylol  on  a  piece  of 
lens  paper.  Immediately  afterward  the  lens  should  be  dried.  Dried  oil  on  a  lens 
often  causes  the  lens  to  be  considered  defective.  Accidental  contact  of  the  dry 
objectives  with  oil  is  not  uncommon  and  should  always  be  thought  of  when  satis- 
factory optical  effects  are  not  obtainable.  In  depositing  the  drop  of  immersion  oil 
on  the  slide  bubbles  are  at  times  formed  which  make  it  almost  impossible  to  use  the 
H  2-inch  objective.  Under  such  circumstances  I  either  prick  the  bubbles  or  wipe  off 
the  oil  and  deposit  a  drop  anew. 

It  is  advisable  to  cultivate  the  use  of  both  eyes  in  doing  microscopical  work. 
When  using  one  eye  the  other  should  be  kept  open  with  accommodation  relaxed. 
It  is  this  squinting  of  the  unemployed  eye  which  so  often  fatigues.  A  strip  of  card- 


ILLUMINATION  5 

board  4  or  5  inches  long,  with  an  opening  to  fit  over  the  tube  of  the  microscope, 
leaving  the  other  end  to  block  the  vision  of  the  unused  eye,  will  prevent  the  strain. 
This  apparatus  can  be  purchased  in  vulcanite. 

Warm  Stages. — A  warm  stage  for  the  study  of  living  protozoa  may  be  extempor- 
ized by  taking  a  piece  of  copper  about  the  size  of  the  stage  and  with  a  strip  projecting 
out  anteriorly  for  5  or  6  inches.  The  under  surface  of  the  plate  is  covered  with 
flannel  and  a  hole  about  i  inch  in  diameter  cut  out  of  the  center.  The  proper  amount 
of  heat  is  applied  by  a  flame  impinging  on  the  tongue-like  projection  of  the  copper 
plate. 

At  present  there  are  electrically  heated  warm  stages,  connected  with  the  desk 
socket  by  a  wire  and  plug,  which  are  most  convenient.  In  fact  they  are  almost  as 
satisfactory  as  the  more  expensive  and  less  convenient  warm  chamber  or  microscope 
oven  surrounding  the  microscope. 

Illumination. — Direct  sunlight  or  excessively  bright  light  is  to  be 
avoided.  If  such  conditions  must  exist  a  white  shade  or  muslin  curtain 
drawn  across  the  window  is  a  necessity.  Light  from  the  north  and  from 
a  white  cloud  is  the  most  desirable.  South  of  the  equator  a  southern 
light.  In  the  tropics  a  piece  of  plate  glass  fitted  into  the  lower  part  of 
a  wire  screen  frame  gives  good  lighting,  keeps  out  dust,  and  does  not 
interfere  greatly  with  the  circulation  of  the  air. 

The  technic  in  connection  with  proper  illumination  is  probably  more  important 
than  any  other  point;  unless  the  light  is  utilized  to  the  best  advantage,  the  best 
results  cannot  be  obtained.  In  examining  fresh  blood  preparations  or  hanging  drops 
the  concave  mirror  should  be  used  and  the  light  almost  shut  off  by  the  iris  dia- 
phragm so  as  to  give  a  contour  picture.  In  examining  a  stained  blood  or  bacterial 
preparation,  the  Abbe  condenser  should  be  properly  focused  so  as  to  best  illuminate 
the  stained  film.  In  many  instruments  set-screws  are  provided  which  check  the  ele- 
vation of  the  Abbe  condenser  when  the  proper  focus  is  reached.  Inasmuch  as  the 
light  from  the  condenser  should  come  to  a  focus  exactly  level  with  the  object  studied, 
it  is  evident  that  a  fixed  position  for  the  condenser  would  not  answer  when  slides  of 
different  thickness  were  used.  Always  use  the  plane  mirror  when  examining  stained 
bacterial  or  blood  films,  as  a  color  image  is  desired.  Ordinarily  in  examining  tissue 
sections,  the  Abbe  condenser  should  either  be  put  out  of  focus  by  racking  down  or  by 
the  use  of  the  concave  mirror  and  the  narrowing  of  the  aperture  of  the  iris  diaphragm. 
Swing-out  condensers  are  now  made  which  are  very  convenient.  The  proper  em- 
ployment of  illumination  only  comes  with  experience,  and  one  should  continue  to 
manipulate  his  mirrors,  diaphragm,  and  condenser  until  the  best  result  is  obtained. 
Then  study  the  specimen. 

For  microscopical  work  in  a  laboratory  not  properly  supplied  with  windows  or  for 
night  work  the  frosted  incandescent  bulb  is  very  satisfactory. 

An  objection  to  artificial  light  is  that  one  working  almost  entirely 
with  sunlight  forms  standards  and  when  using  a  different  light  is  some- 
what confused  in  interpretation  of  the  microscopical  picture. 


6  APPARATUS 

There  is  now  on  the  market  a  special  electric  lamp  and  light  filter  which  tends  to 
give  optical  effects  similar  to  that  obtained  with  sunlight.  These  are  called  daylight 
filters.  The  bulb  of  the  lamp  is  filled  with  nitrogen  gas  and  the  glass  filter  screen  has 
a  bluish  color.  By  using  a  6-inch  round-bottom  flask  filled  with  an  alkaline  solution 
of  copper  sulphate  as  a  condenser,  similar  illuminating  effect  to  that  of  the  day- 
light lamp  can  be  obtained. 

Dark  Ground  Illumination. — Very  valuable  information,  especially 
as  regards  the  detection  of  treponemata  in  material  from  hard  chancres 
or  mucous  patches,  may  be  obtained  by  the  use  of  dark  ground  illumina- 
tion. There  are  many  different  types  of  apparatus  for  this  purpose. 

The  bacteria  or  spirochaetes  are  intensely  illuminated  and  show  as 
brilliant  silvery  objects  in  contrast  to  the  dark  background. 

When  the  morphological  details  of  a  brightly  illuminated  object  in  the  dark  field 
can  be  distinctly  observed  it  is  proper  to  use  the  term  dark  ground  illumination. 
When  only  particles,  usually  surrounded  by  bright  and  dark  rings,  and  not  showing 
any  structure,  are  observed  in  the  dark  field  the  proper  designation  is  ultramicro- 
scopic.  An  apparatus  using  only  the  short  waves  of  the  ultra-violet  spectrum 
enables  one  to  observe  particles  no  larger  than  Ho  of  a  micron.  For  this  apparatus 
it  is  necessary  to  employ  photographic  plates.  In  using  the  H  2-inch  objective  with 
dark  ground  illumination  a  funnel-like  base  is  supplied  on  which  we  screw  the  nickle- 
plated  front  mount  of  the  objective.  Before  using  the  dark-field  apparatus  it  must 
be  centered  with  a  low  power.  This  is  carried  out  by  getting  concentric  rings  paral- 
lel with  the  circle  of  the  microscopic  field.  Immersion  contact  between  the  front 
surface  of  the  Abbe  condenser  and  the  under  surface  of  the  slide  carrying  the  prepa- 
ration must  be  made  before  focussing  the  ^2th  objective.  As  a  source  of  illu- 
mination we  may  use  a  small  arc-lamp  or  a  Nernst  lamp  or  an  incandescent  gas  lamp. 
In  using  an  arc-lamp  one  must  have  a  suitable  rheostat  according  to  the  electrical 
current  employed.  Information  as  to  voltage  and  nature  of  current  must  be  given 
the  one  supplying  the  apparatus. 

In  making  preparations  the  slides  and  cover-slips  should  be  scrupulously  clean 
and  the  material  thinly  spread  out  and  free  of  bubbles.  With  low  power  objectives 
one  can  obtain  satisfactory  dark-field  illumination  by  pasting  a  circle  of.black  paper 
in  the  center  of  one  of  the  glass  discs  which  fit  in  the  ring  under  the  lens  of  the  sub- 
stage  condenser.  The  diameter  of  the  opaque  center  will  have  to  be  greater  as  the 
magnifying  power  of  the  objective  increases  and  for  oil-immersion  objectives  a  special 
apparatus  is  required.  Flagellates  in  faeces  are  best  studied  with  the  dark-field 
illumination. 

.APPARATUS  FOR  STERILIZATION 

For  the  purpose  of  sterilizing  glassware,  media,  and  old  cultures  there 
are  three  methods  ordinarily  employed.  Th«  hot-air  sterilizer,  in  which 
a  temperature  of  about  i5o°C.  is  maintained  for  one  hour,  is  ordinarily 
used  for  the  sterilization  of  Petri  dishes,  test-tubes,  pipettes,  etc. 


STERILIZERS  7 

If  the  temperature  is  allowed  to  go  too  high,  there  is  danger  of  charring  the  cotton 
plugs  and  also  of  causing  the  development  of  an  empyreumatic  oil  which  makes  the 
plugs  unsightly  and  causes  them  to  stick  to  the  glass.  Again  we  must  be  careful  not 
to  open  the  door  until  the  temperature  has  fallen  to  6o°C.,  otherwise  there  is  danger 
of  cracking  the  glassware.  Where  gas  is  not  obtainable,  the  hot-air  sterilizer  is  not  a 
very  satisfactory  apparatus. 




FIG.  i.— Dressing  sterilizer  showing  cylinder  containing  water  (K)  heated  either 
by  gas  or  Primus  kerosene  lamps. 

Arnold  Sterilizer.— The  Arnold  sterilizer  is  to  be  found  everywhere 
and  can  be  used  on  blue-flame  kerosene-oil  stoves  as  readily  as  with  gas 
burners.  The  most  convenient  form,  but  more  expensive,  is  the  Boston 
Board  of  Health  pattern.  The  ordinary  pattern,  with  a  telescoping 
outer  portion,  answers  all  purposes,  however. 


8  APPARATUS 

In  the  Arnold,  sterilization  is  effected  by  streaming  steam  at  ioo°C.  It  is  usual  to 
maintain  this  temperature  for  fifteen  to  twenty-five  minutes  each  day  for  three  suc- 
cessive days.  The  success  of  this  procedure — fractional  sterilization —  is  due  to  the 
fact  that  many  spores  which  were  not  killed  at  the  first  steaming  have  developed  into 
vegetative  forms  within  twenty-four  hours,  and  when  the  steam  is  then  applied  such 
forms  are  destroyed.  Experience  has  shown  that  all  the  spores  have  developed  by 
the  time  of  the  third  steaming,  so  that  with  this  final  application  of  heat  we  secure 
perfect  sterilization. 

It  is  customary  to  use  the  Arnold  for  sterilizing  gelatin  and  milk 
media,  even  when  the  autoclave  is  at  hand,  the  idea  being  that  the 
greater  heat  of  the  autoclave  may  interfere  with  the  quality  of  such 
media.  The  most  convenient  autoclave  is  the  horizontal  type,  such  as 
is  to  be  found  everywhere  for  the  sterilization  of  surgical  dressings. 

The  source  of  heat  may  be  either  electricity,  gas,  the  Primus  kerosene-oil  lamp  or 
steam  from  an  adjacent  boiler.  More  recently  a  method  of  employing  kerosene,  gaso- 
lene, or  alcohol  with  a  gravity  system  has  been  perfected.  During  the  past  ten  years, 
in  the  laboratory  of  the  U.  S.  Naval  Medical  School,  we  have  been  using  a  dressing 
sterilizer,  made  by  the  American  Sterilizer  Co.,  with  which  it  has  been  possible  to 
most  satisfactorily  carry  out  all  kinds  of  sterilization,  thus  doing  away  with  the  use 
of  the  Arnold  and  the  hot-air  sterilizer.  It  is  impossible  to  sterilize  ordinary  fermen- 
tation tubes  in  the  autoclave  on  account  of  the  boiling  up  of  the  media  and  wetting 
of  the  plugs.  This  is  still  done  with  the  Arnold.  By  use  of  the  Durham  tubes — 
which  are  to  be  preferred,  except  for  gas  analysis — sugar  media  can  be  thus  sterilized. 

Should  a  small  bubble  remain  in  the  top  of  the  small  inverted  inner  tube  after 
removal  from  the  autoclave,  one  may  make  a  mark  with  a  grease  pencil  at  the  line 
of  the  bubble;  or,  if  preferred,  the  basket  of  Durham  tubes  can  be  heated  to  boiling 
for  ten  minutes  in  a  pan  of  water  or  in  the  Arnold  when,  after  cooling,  the  bubble  will 
be  found  to  have  disappeared. 

Glassware  will  come  out  from  such  an  autoclave  with  wrappers  as  dry  and  plugs 
of  the  test-tubes  as  stopper-like  as  could  be  effected  in  a  hot-air  sterilizer. 

The  objection  which  exists  in  the  use  of  some  autoclaves,  as  regards  condensa- 
tion on  dressings  or  apparatus,  does  not  exist  in  this  type.  The  mechanism,  by 
which  the  inner  and  outer  chambers  are  connected  and  disconnected,  and  that  for 
vacuum  production,  rests  in  the  simple  turning  of  a  lever  from  mark  to  mark.  We 
have  been  able  with  a  gas  burner  to  obtain  a  pressure  of  15  pounds  in  less  than  ten 
minutes.  In  sterilizing  test-tubes  we  place  them  in  small  rectangular  wire  baskets, 
6X5X4  inches.  These  baskets  are  to  be  preferred  to  round  ones,  as  they  pack  more 
satisfactorily  in  the  refrigerator  used  for  storing  media.  In  sterilizing  flasks,  test- 
tubes,  Petri  dishes,  throat  swabs,  pipettes,  etc.,  it  has  been  our  custom,  after  ex- 
posing to  20  pounds'  pressure  for  twenty  minutes,  to  produce  a  vacuum  for  two  or 
three  minutes;  then  with  the  steam  in  the  outer  jacket  for  a  few  minutes  to  thor- 
oughly dry  the  articles  in  the  disinfecting  chamber.  The  valve  to  the  inner  chamber 
is  then  opened  to  break  the  vacuum;  the  door  is  now  opened,  and  the  articles  re- 
moved in  as  dry  a  state  as  if  they  had  been  in  the  hot-air  sterilizer.  Articles,  how- 
ever, can  be  thoroughly  dried  without  the  use  of  a  vacuum,  simply  allowing  the  steam 
to  remain  in  the  outer  jacket  with  the  steam  cut  off  from  the  inner  chamber. 


CLEANING  GLASSWARE  9 

PRESSURE  AND  TEMPERATURE  TABLE 

5  pounds' pressure,  107. 7°C.,  226°F. 

10  pounds' pressure,  115. 5°C.,  24o°F. 

15  pounds' pressure,  121. 6°C.,  25o°F. 

20  pounds' pressure,  i26.6°C.,  26o°F. 

25  pounds' pressure,  130. 5°C.,  267°F. 

30  pounds' pressure,  134. 4°C.,  274°F. 

All  such  articles  as  Petri  dishes,  pipettes,  swabs,  etc.,  are  wrapped  in  cheap 
quality  filter-paper,  making  a  fold  and  turning  in  the  ends  as  is  done  in  a  druggist's 
package.  Old  newspapers  answer  well  for  this  purpose.  The  sterile  swab  can  be 
used  for  many  purposes  in  the  laboratory.  They  are  most  easily  made  by  taking 
a  piece  of  copper  wire  about  8  inches  long,  flattening  one  end  with  a  stroke  of  a 
hammer,  then  twisting  a  small  pledget  of  plain  absorbent  cotton  around  the  flat- 
tened end.  After  wrapping,  the  swabs  are  sterilized  in  bunches.  We  not  only  use 
them  for  getting  throat  cultures,  but  in  addition  for  culturing  faeces,  pus,  or  other 
such  material.  The  pus  is  obtained  with  a  swab,  which  material  is  then  distributed 
in  a  tube  of  sterile  bouillon  or  water.  With  the  same  swab  the  surface  of  an  agar 
plate  is  successively  stroked.  This  method  is  almost  as  satisfactory  as  the  German 
one  of  using  bent  glass  rods  for  this  purpose.  Everyone  has  encountered  the  diffi- 
culties attendant  upon  the  bending  of  platinum  wires  and  also  the  possibility  of 
destroying  your  organisms  by  an  insufficiently  cooled  wire. 


CLEANING  GLASSWARE 

It  is  a  routine  in  our  laboratory  for  everything  to  go  through  the 
sterilizer  at  i25°C.  before  anything  else  is  done.  This  is  a  safe  rule 
when  dealing  with  dangerous  pathogenic  organisms  (especially  tetanus 
and  anthrax). 

As  soon  as  taken  out  of  the  sterilizer  the  contents  are  emptied,  and  the  tube  or 
dishes  placed  in  a  i  %  solution  of  washing  soda  and  boiled.  This  thoroughly  cleans 
them.  As  the  washing  soda  slightly  raises  the  boiling-point  and  also  makes  the 
spores  more  penetrable,  it  would  appear  that  under  ordinary  circumstances,  it  would 
be  sufficient  to  place  all  contaminated  articles  in  a  dishpan  with  the  soda  solution, 
and  boil  for  at  least  one  hour,  not  using  a  preliminary  sterilization  in  the  autoclave. 
The  tubes  are  now  cleaned  with  a  test-tube  brush,  thoroughly  rinsed  with  tap  water 
and  placed  in  a  i%  solution  of  hydrochloric  acid  for  a  few  minutes;  then  rinsed 
thoroughly  in  water  and  placed  in  test-tube  baskets,  mouth  downward,  and  allowed 
to  drain  over  night.  Some  laboratory  workers  boil  their  test-tubes  and  other  glass- 
ware in  water  containing  soap  or  soap  powder  and,  after  a  thorough  rinsing  in  tap 
water,  drain.  Hydrochloric  acid  should  not  be  used  after  the  soap  as  it  will  cause 
the  formation  of  an  unsightly  coating  difficult  to  remove.  When  thoroughly  dry 
they  may  be  plugged  and  sterilized.  To  plug  a  test-tube,  pick  out  a  little  pledget  of 
plain  absorbent  cotton  about  2  inches  in  diameter  from  a  roll.  Place  it  over  the  cen- 
ter of  the  tube  and  with  a  glass  rod  push  the  cotton  down  the  tube  about  an  inch.  In 


IO 


APPARATUS 


culturing  slow  growing  organisms,  as  tubercle  bacilli,  or  certain  pathogenic  protozoa 
it  is  necessary  to  have  plugs  so  prepared  as  to  prevent  drying  out  of  the  medium  in  the 
tube.  The  simplest  way  of  accomplishing  this  is  to  melt  some  paraffin  in  a  pan,  then 
removing  the  cotton  plug  with  the  fingers  to  dip  the  end  entering  the  tube  into  the 
melted  paraffin  and  then  push  the  surface  so  prepared  into  the  tube.  Plasticine, 
sealing  wax  or  paraffin  may  be  used  to  seal  over  the  tops  of  such  test-tubes. 

Cleaning  Fluid. — The  cleaning  fluid  commonly  used  in  laboratories  consists  of  i 
part  each  of  potassium  bichromate  and  commercial  sulphuric  acid  with  10  parts  of 
water.  This  is  an  excellent  mixture  for  cleaning  old  slides,  etc.,  especially  when 
grease  or  balsam  is  to  be  gotten  rid  of.  It  is  very  corrosive,  however. 


FIG.  2. — i,  Inoculation  of  tubes;  2,  plugging  of  tubes;  3,  filling  tubes;  4,  Smith's 
fermentation  tube;  5,  Durham's  fermentation  tube. 


In  cleaning  glassware  for  such  tests  as  the  colloidal-gold  one  it  is  essential  that  we 
use  such  a  cleaning  fluid.  An  efficient  and  less  corrosive  method  for  cleansing  slides 
and  cover-glasses  is  to  leave  them  over  night  in  an  acetic  acid  alcohol  mixture  (two 
parts  of  glacial  acetic  acid  to  100  parts  of  alcohol).  After  drying  and  polishing  out 
of  this  mixture,  it  is  well  to  pass  the  slides  and  cover-glasses  through  the  flame  of 
a  Bunsen  burner  or  alcohol  lamp  to  remove  every  vestige  of  grease.  Ordinarily, 
rubbing  between  the  thumb  and  forefinger  with  soap  and  water,  then  drying  with 
an  old  piece  of  linen,  and  finally  flaming  will  yield  a  perfect  surface  for  making  a 
bacterial  preparation. 


HANGING  DROP  II 

CONCAVE  SLIDES,  FERMENTATION  TUBES 

The  concave  slide  is  ordinarily  used  for  making  hanging-drop  prepa- 
rations for  the  examination  of  bacteria  as  to  motility,  capsules,  size 
and  arrangement. 

To  prepare  a  hanging-drop  preparation  for  the  study  of  motility  it  is  best  to  place 
a  loopful  of  the  young  bouillon  culture  or  a  loopful  of  salt  solution  into  which  is  then 
emulsified  a  small  amount  of  growth  from  an  agar  slant,  in  the  center  of  the  cover- 
glass;  now  having  applied  with  a  brush  a  ring  of  vaseline  around  the  concave  depres- 
sion in  the  slide  we  apply  the  slide  as  a  cover  to  the  cover-glass  which  latter  adheres 
to  the  ring  of  vaseline.  The  completed  hanging-drop  preparation  can  now  be  turned 
over  and  placed  on  the  stage  of  the  microscope. 

A  substitute  which  is  equally  good  may  be  made  by  spreading  a  ring  or  square  of 
vaseline — smaller  than  the  cover-glass  to  be  used — in  the  middle  of  a  plain  slide. 
Then  putting  a  loopful  of  salt  solution  in  the  center  of  the  space,  and  inoculating  with 
the  culture  to  be  studied,  we  finally  cover  it  with  a  cover-glass,  gently  pressing  the 
margins  down  on  the  vaseline.  This  gives  a  preparation  for  the  study  of  motility  or 
agglutination  which  does  not  dry  out  for  hours,  and  is  easier  to  focus  upon  than  the 
concave  slide  hanging-drop  preparation. 

Hanging  Drop. — In  examining  a  hanging  drop  first  use  a  low- 
power  objective  and,  having  brought  into  focus  the  margin  of  the  drop 


FIG.  3. — Hanging  drop,  over  hollow  ground  slide.     (Mac  Neal.)     » 

as  a  center  line,  change  to  a  J£-  or  J^-inch  objective.  By  this  pro- 
cedure a  thin  layer  of  fluid  is  brought  under  the  high  dry  objective 
instead  of  the  deeper  layer  in  the  center  of  the  drop.  It  is  not  ad- 
visable to  use  an  immersion  objective  with  a  hanging-drop  preparation. 

The  light  should  be  cut  down  to  a  minimum  with  the  iris  diaphragm  and  the  con- 
cave mirror  used.  When  we  have  finished  examining  the  preparation  the  cover-glass 
should  be  pushed  over  with  the  forceps  so  that  a  corner  projects  and  we  then  seize 
this  with  the  forceps,  lift  up  the  cover-glass  and  drop  it  into  the  disinfecting  solu- 
tion along  with  the  slide. 

Fermentation  Tubes. — The  fermentation  tube  with  a  bulb  and  closed 
arm  is  expensive,  difficult  to  clean,  and  is  easily  broken.  It  is,  however, 
convenient  in  the  determination  of  the  gas  formula  of  an  organism. 
Its  use  is  described  under  water  analysis.  As  a  substitute  in  the  study 
of  gas  production  and  in  water  bacteriology,  the  Durham  tube  is  to  be 
recommended. 


12 


APPARATUS 


Into  a  test-tube,  about  1X7  inches,  we  introduce  the  special  sugar  media,  then 
drop  down  a -small  test-tube  (KX3  inches)  with  its  open  end  downward.  Insert 
the  plug  of  the  large  tube  and  sterilize.  During  sterilization  the  fluid  enters  the 
mouth  of  the  smaller  tube  and  fills  it,  and  when  the  medium  is  subsequently  inocu- 
lated, if  gas  forms,  it  appears  in  the  upper  part  of  the  closed  end  of  the  smaller  tube. 


FIG.  4. — Blood  serum  coagulating  apparatus. 

Inspissators. — For  inspissating  blood-serum  slants  a  regular  inspissa- 
tor  is  desirable. 

This  is  nothing  more  than  a  double-walled  vessel,  the  space  between 
the  walls  being  filled  with  water. 

As  a^  substitute  one  may  take  the  common  rice  cooker  (double  boiler).  Fill  the 
outer  part  with  water;  and  in  the  inner  compartment  pack  the  serum  tubes  properly 

slanted  on  a  piece  of  wood  or  a  wedge-shaped 
layer  of  cotton.  Place  a  weight  on  the  cover 
of  the  inner  compartment  to  sink  it  into  the 
surrounding  water,  and  allow  to  boil  for  one 
or  two  hours.  This  same  apparatus  may  be 
used  for  their  sterilization  on  two  subsequent 
days,  but  it  is  better  to  sterilize  in  the  auto- 
clave or  Arnold.  The  rice  cooker  is  of  the 
greatest  use  in  preparing  culture  media.  For 
this  purpose  the  outer  compartment  is  filled 
with  calcium  chloride  solution  or  a  25%  solu- 
tion of  common  salt,  so  that  the  tempera- 
ture of  the  contents  of  the  inner  receptacle  may  be  raised  to  the  boiling  point. 
Of  course  media  may  be  prepared  in  an  ordinary  sauce-pan  but  there  is  great 
danger  of  scorching  media  prepared  in  this  way.  Special  vessels  with  two  bottoms, 
between  which  is  an  air  space,  are  now  on  the  market  and  have  an  advantage 
over  the  double  boiler  (rice  cooker). 

Ebony  Finish. — As  regards  a  working  desk,  it  will  be  found  convenient  to  have  an 
arrangement  similar  to  the  ordinary  flat-top  desk,  with  a  tier  of  drawers  on  each  side. 


FIG.  5.— Rice  cooker. 


EBONY  FINISH  13 

A  block  of  wood  with  holes  bored  in  it  to  contain  dropping-bottles  may  be  placed  in 
the  upper  left-hand  drawer.  In  this  way  the  stains  are  as  accessible  as  if  they  encum- 
bered the  desk.  It  is  advisable  to  paint  the  inside  of  this  drawer  black  so  that  the 
light  may  not  cause  the  staining  reagents  to  deteriorate. 

A  very  popular  method  of  preparing  the  wooden  surfaces  of  labora- 
tory desks,  sinks,  and  tables  is  the  application  of  the  so-called  "acid- 
proofing."  This  gives  an  ebony-like  finish  which  is  not  affected  by 
strong  acids. 

In  using  it  the  surface  of  the  wood  must  be  new  (free  of  any  varnish,  oil,  or  paint, 
if  previously  so  coated  the  surface  must  be  planed). 

Solution  i 

Potassium  chlorate 125  .o  grams. 

Cupric  sulphate 125 .  o  grams. 

Water 1000 .  o  c.c. 

Apply  two  coats  of  this  solution  at  least  twelve  hours  between  applications. 
When  thoroughly  dry  apply  two  coats  of  solution  No.  2. 

Solution  2 

Aniline  oil 120.0  c.c. 

Hydrochloric  acid 180.0  c.c. 

Water 1000.0  c.c. 

When  the  treated  surface  is  thoroughly  dry  apply  one  coat  of  raw  linseed  oil  with  a 
cloth.  After  this  is  dry  wash  with  very  hot  soapsuds. 

An  aspirating  bottle  on  a  shelf  elevated  2  feet,  with  rubber  tubing  and  glass 
tip  leading  to  a  small  aquarium  jar  or  other  desk  receptacle,  makes  a  good  substitute 
for  a  small  sink  and  faucet.  A  Hofmann  screw  clamp  on  the  rubber  tube  controls 
the  flow  of  water. 

Ordinary  glass  salt  cellars  will  be  found  very  useful,  where  the  watch-glass  is 
employed.  They  may  also  be  wrapped,  sterilized,  and  used  to  contain  fluids  for 
inoculating,  etc. 

A  glass-topped  fruit  jar  or  a  specimen  jar  containing  a  disinfecting  solution  for  con- 
taminated slides,  etc.,  should  be  on  every  working  desk.  A  good  solution  is  that  of 
Harrington  (corrosive  sublimate, -0.8;  commercial  HC1,  60.0  c.c.;  alcohol,  400.0 
c.c.;  water,  to  1000.0  c.c.). 

Disinfectant  Solution. — A  very  simple  method  of  making  a  disinfec- 
tant similar  to  lysol  is  to  put  one  part  of  cresol  or  crude  carbolic  acid 
and  one  part  of  soft  soap  in  a  wide-mouthed  bottle  over  night.  The 
resulting  compound  (Liquor  cresolis  comp.  U.  S.  P.)  makes  a  perfect 
solution  with  water  and  a  5%  solution  of  this  will  be  found  at  least 
equal jto  a  5%  phenol  solution.  In  addition  to  using  as  a  desk  jar  dis- 
infectant it  is  excellent  for  disinfecting  faeces,  sputum,  etc. 


14  APPARATUS 

Platinum  Loops.— For  use  in  making  loops  and  needles,  platinum  wire  of  26  gauge 
will  be  found  most  suitable.  The  handle  made  of  glass  rod  is  preferable  to  the 
metal  ones.  One  end  is  fused  in  the  flame  and,  holding  the  3-  to  4-in.  piece  of  plati- 
num wire,  with  forceps,  in  the  same  flame,  insert  the  glowing  metal  into  the  molten 
glass.  By  taking  two  lengths  of  platinum  wire  and  twisting  them  together  a  more 
rigid  needle  is  made  for  inoculating  stab  cultures. 


Isolation   cf  colonies    uslny  two  halves  cf 
ayar  plate    Instead   cf  two   separate  plates. 


Zlnsser's     anaerobic 
plate,    method 


gutter's     vaccine 
ampule  filler 


FIG.  6. — i,  Method  of  using  one  plate  instead  of  two  for  isolation  of  colonies, 
(see  page  50).  The  separated  colonies  on  the  No.  II  side  of  the  plate  are  studied 
with  unaided  eye  and  aplanatic  triplet  (^  in.  or  %  in.)  both  by  reflected  and  trans- 
mitted light.  After  we  have  determined  the  presence  of  two  or  more  different 
kinds  of  colonies,  a  well  separated  one  of  each  type  is  selected  and  a  blue  pencil  ring 
made  around  it  on  the  glass  surface  of  the  back  of  the  Petri  dish  together  with  a  num- 
ber. This  number  is  carried  along  on  culture  tubes  or  microscopical  slide  prepara- 
tions until  the  organism  is  identified.  2,  Lyon  tube  for  blood.  More  convenient 
than  the  Wright  tube.  For  description  see  page  18.  3,  Bronfenbrenner's  an- 
aerobic tube.  This  is  made  by  drawing  out  a  single  test-tube  instead  of  using  two 
separate  tubes  as  with  the  Noguchi  method.  Before  drawing  out  the  test-tube  the 

Eiece  of  sterile  tissue  is  introduced  and  after  drawing  out  in  the  flame  the  ascitic 
roth  or  sheep  serum  water,  as  well  as  the  material  for  culturing,  is  introduced 
through  the  drawn-out  neck  with  a  bulb  capillary  pipette.  The  lower  part  of  the 
tube  is  placed  in  water  at  37°C.  and  the  vacuum  connection  made  and  after  exhaus- 
tion of  air  the  sterile  paraffin  oil  is  run  in.  The  drawn-out  portion  is  then  sealed  off 
in  a  small  flame  and  looks  like  a  sealed  vaccine  ampule.  4,  Noguchi  apparatus. 
5,  Zinsser  method  for  anaerobic  plates  (page  80).  6,  Butler's  apparatus  for  filling 
vaccine  ampules.  The  sterile  vaccine  is  put  in  the  sterile  flask,  and  the  stopper  with 
air  intake  and  needle  filler  separately  sterilized  and  then  introduced  into  neck  of 
flask  which  is  then  inverted. 


A  platinum  loop  made  around  a  piece  of  copper  wire,  4  mm.  in  diameter  holds 
about  2  mg.  of  culture  taken  up  from  an  agar  slant.  Kolle  estimates  that  an 
agar  slant  of  typhoid  bacilli  or  of  staphylococci  should  contain  15  such  loopfuls 


INCUBATORS  1 5 

while  a  streptococcus  slant  would  have  almost  five.  It  has  been  estimated  that  such 
a  standard  loop  would  contain  between  2,000,000,000  and  3,000,000,000  organisms. 
Of  the  greatest  use  in  culturing  material  obtained  at  autopsy  is  the  platinum  spud. 
This  can  be  made  by  hammering  out  one  end  of  a  piece  of  15-  to  i8-gauge  platinum 
wire. 

For  making  smears  from  faeces,  sputum,  and  the  like,  wooden  tooth- 
picks are  very  convenient;  the  kind  with  the  spatulate  end  is  preferable. 

Incubators. — When  gas  is  obtainable,  the  maintaining  of  a  constant  temperature 
for  the  body  temperature  incubator  (38°C.)  and  the  paraffin  oven  (6o°C.)  is  best 
secured  by  the  use  of  some  of  the  various  types  of  thermo-regulators.  The  Reichert 
type  is  the  one  in  general  use,  although  there  are  many  features  about  the  Dunham 
and  Roux  regulators  which  are  advantageous.  • 

If  the  pressure  of  the  gas-supply  varies  from  time  to  time,  it  is  essential  to  regulate 
this  by  the  use  of  a  gas-pressure  regulator  (MurrilPs  is  a  cheap  and  satisfactory 
one). 

Incubators,  controlled  electrically,  can  be  obtained  of  certain  foreign  makers,  and 
are  quoted  in  catalogues  of  American  dealers.  It  is  probable  that  the  Koch  petro- 
leum lamp  incubator  is  the  most  satisfactory  one  where  gas  is  not  obtainable. 
They  should  be  of  all  metal  construction,  and  not  with  a  wood  casing,  on  account  of 
the  danger  from  fire.  They  cost  from  twenty-five  to  fifty  dollars. 

An  incubator  may  be  extemporized  by  putting  the  bulb  of  an  incandescent  electric 
lamp  in  a  vessel  of  water.  The  proper  temperature  may  be  obtained  by  increasing 
the  amount  of  water  or  by  covering  the  opening  more  or  less  completely  with  a  towel. 
The  test-tubes  to  be  incubated  can  be  put  into  a  fruit  jar  or  tin  can,  which  receptacle 
is  placed  in  the  vessel  heated  by  the  lamp. 

Emery  suggests  the  use  of  a  Thermos  bottle  as  an  incubator. 

The  vacuum  bottle  should  be  first  warmed  by  pouring  in  warm  water.  After- 
ward the  bottle  should  be  three-fourths  filled  with  water  at  ioo°F. 

Schrup  suspends  his  cultures  and  thermometer  in  the  water  by  threads  attached 
to  pins  in  the  cork  of  the  vacuum  bottle.  The  plug  should  be  paraffined  or  covered 
with  a  rubber  cap.  As  regards  the  matter  of  a  low-temperature  incubator  (for 
gelatin  work),  this  may  be  met  by  using  a  small  refrigerator.  The  ice  in  the  upper 
part  maintains  an  even  cold,  and  by  connecting  up  an  electric  lamp  in  the  lower  part 
of  the  refrigerator  we  can  easily  maintain  a  temperature  which  only  varies  one  or  two 
degrees  during  the  twenty-four  hours.  The  gelatin  plates  or  tubes  should  be  placed 
on  the  shelves  usually  provided  with  the  refrigerator  and  not  on  the  bottom. 

With  a  i6-candle-power  lamp  a  temperature  of  about  2S°C.  is  maintained  (this  is 
too  high,  being  about  the  melting-point  of  gelatin);  with  an  8-candle-power,  one 
about  21°  to  23°C.;  and  with  a  4-candle-power,  from  18°  to  2o°C.;  the  box  being 
about  20X30X36  inches. 

More  recently  we  have  used  with  entire  satisfaction  a  low  tempera- 
ture incubator  made  by  Hearson. 

The  low  temperature  is  supplied  by  water  from  cracked  ice  packed  in  a  large  cen- 
tral chamber.  A  small  dynamo  controlled  by  a  thermostat  circulates  the  water 


i6 


APPARATUS 


around  the  chamber  containing  the  gelatin  cultures.  It  requires  some  time  for 
proper  adjustment,  but  afterward  maintains  a  uniform  temperature.  Should  the 
temperature  of  the  room  in  which  the  incubator  is  installed  be  below  22°C.  there  is 
provided  an  automatically  controlled  heating  coil  which  operates  when  the  surround- 
ing temperature  is  lower  than  22°C. 

Centrifuge. — When  much  serum  reaction  work  is  done,  an  electrically  run  centri- 
fuge is  an  absolute  necessity.  It  should  be  strongly  constructed  and  placed  on  a 
firm  base.  There  should  be  places  for  8  tubes  and  the  outer  shell  or  guard  should 


FIG.  7. — i,  2,  3,  Drawing  put  glass  tubing;  4,  5,  Wright's  rubber  bulb  capillary 
pipettes  showing  grease  pencil  mark  for  making  dilutions;  6,  7,  Wright's  U-tubes; 
8,  p,  10,  methods  of  drawing  out  test-tubes  for  vaccines  in  opsonic  workj'n,  bac- 
teriological pipette. 

be  so  strong  that  in  event  of  the  breaking  of  a  tube,  while  the  centrifuge  is  re- 
volving at  high  speed,  there  would  be  no  danger  for  the  operator.  Water-power- 
driven  centrifuges  are  less  satisfactory  and  hand  ones  least  so. 

An  electric  drying  oven  is  very  useful,  taking  the  place  of  a  water 
bath.  In  reactions  like  that  for  blood  acidosis,  where  the  steam  vapor 
interferes,  it  is  almost  essential. 

Filter  Pump.— A  filter  pump  attached  to  the  water  faucet,  preferably  by  screw 
threads,  is  almost  indispensable  for  filtering  cultures,  etc.,  and  for  cleaning  small 
pipettes,  especially  the  haemocytometer  pipettes.  Such  a  filter  or  vacuum  pump  with 


PIPETTES  17 

a  vacuum  gauge  is  more  easily  controlled.  In  washing  red  cells  in  Wassermann 
reactions  a  pipette  attached  to  the  rubber  tubing  of  the  pump  facilitates  the  removal 
of  the  supernatant  fluid. 

The  filter  pump  is  indispensable  when  using  the  various  types  of  porcelain  or 
Berkefeld  filters.  The  Punkal  or  Muencke  types  of  filter  are  the  most  convenient 
in  filtering  toxins  or  in  the  sterilization  of  certain  media  when  heating  would  be 
unadvisable. 


-L-AVERY- 


FIG.  8. — i,  Apparatus  combining  various  methods  for  culture  of  anaerobes;  (a) 
Hofmann  clamp  for  connecting  with  vacuum  pump;  (6)  pyrogallic  at  bottom  of 
bottle  for  Buchner's  O  absorption  method;  (c)  deep  glucose  agar  stab  covered  with 
sterile  liquid  petrolatum  (see  anaerobes).  2,  One-fourth  inch  capillary  loop  U  tube 
for  making  two  nitric  acid  albumin  tests  (see  chemical  examination  of  urine).  3, 
Piece  of  tubing  bent  to  hold  slide  for  steaming  smears  in  flame.  4,  Schmidt's  fer- 
mentation apparatus,  as  modified  by  using  graduated  cylinder  (see  under  faeces). 
5,  One-fourth  inch  glass  tubing,  4^  inches  long  with  ^corks  at  each  end.  For 
centrifuging  faeces  for  ova.  6a,  Apparatus  connected  with  sterile  centrifuge  tube 
for  taking  blood  from  vein  of  man  or  a  guinea-pig  or  rabbit's  heart.  6b,  Erlenmeyer 
flask  which  can  be  used  instead  of  centrifuge  tube.  See  under  sections  Immunity 
and  Blood.  7,  A  graduated  pipette  with  Hofmann  clamp  applied  to  rubber  bulb 
for  precise  delivery  of  measured  quantities  of  liquids. 

Capillary  Pipettes. — With  the  possible  exception  of  the  platinum 
loop,  there  is  no  piece  of  apparatus  so  applicable  to  many  uses  as  the 
capillary  pipette  made  from  a  piece  of  glass  tubing. 

These  may  be  made  in  a  great  variety  of  shapes.    The  one  with  a  hooked  end,  the 
Wright  tube,  is  the  best  apparatus  for  securing  blood  for  serum  tests.     The  crook 
hangs  on  the  centrifuge  guard  and  by  filing  and  breaking  the  thicker  part  of  the  tube 
2 


1  8  APPARATUS 

the  serum  is  accessible  to  a  capillary  rubber  bulb  pipette  or  to  the  tip  of  a  haemocy- 
tometer  pipette.     In  this  way  dilutions  of  serum  are  easily  made. 

Quite  recently  I  have  been  using  the  blood  tube  recommended  by  LYON.  To  make 
it  heat  a  5-  or  6-inch  section  of  H  inch  tubing  in  the  center  and  draw  out  as  for 
making  2  bacteriological  pipettes.  Divide  and  seal  off  the  large  end  in  the  flame. 
Next  seal  off  the  capillary  end.  Then  apply  a  very  small  flame  to  a  point  on  the 
large  end  just  before  it  begins  to  taper  to  the  capillary  part.  The  heat  causes 
the  heated  sealed  off  air  inside  to  force  out  a  blow  hole.  To  use:  Break  off  the 
sealed  capillary  end  and  allow  the  capillary  end  to  suck  up  blood  from  a  drop  just 
as  with  the  Wright  tube.  I  consider  this  tube  superior  to  the  Wright  one. 


•  Pipettes.—  The  capillary  pipette  is  made  by  taking  a  piece  of  J 

soft  German  glass  tubing,  about  6  inches  long,  and  heating  in  the  middle 

in  a  Bunsen  flame,  revolving  the  tubing  while  heating  it. 

When  it  becomes  soft  in  the  center,  remove  from  the  flame  and  with  a  steady  even 
pull  separate  the  two  ends.  The  capillary  portion  should  be  from  1  8  to  20  inches  in 
length.  When  cool,  file  and  break  off  this  capillary  portion  in  the  middle.  We  then 
have  two  capillary  pipettes.  By  using  a  rubber  bulb,  such  as  comes  on  medicine 
droppers,  we  have  a  means  of  sucking  up  and  forcing  out  fluids  by  pressure  with  the 
thumb  and  forefinger  of  the  right  hand.  The  bulb  should  be  pushed  on  about  H 
to  Y±  inch;  this  gives  a  firmer  surface  to  control  the  pressure  on  the  bulb. 

A  bacteriological  pipette  is  made  by  drawing  out  a  g-inch  piece  of  tubing  about 
3  inches  at  either  end,  then  heating  in  the  middle  we  draw  out  and  have  two 
pipettes  similar  to  the  one  shown  in  the  drawing.  A  piece  of  cotton  is  loosely  pushed 
in  just  above  the  narrow  portion.  These  may  be  wrapped  in  paper  and  sterilized  for 
future  use.  They  may  be  made  perfectly  sterile  at  the  time  of  drawing  out. 

Illustrations  are  given  for  apparatus  for  culturing  spirochaetes  as  well  as  for 
anaerobic  plating  methods  in  Fig.  6.  In  the  same  cut  is  shown  an  apparatus  for 
filling  vaccine  ampoules  and  also  our  aerobic  plating  method  for  class  work. 

Where  gas  is  not  at  hand,  the  Barthel  alcohol  lamp  gives  a  flame  similar  to  that 
of  the  Bunsen  lamp  and  is  equally  satisfactory  for  heating  glass  tubing.  By  making 
a  collar  with  a  lateral  opening  to  fit  the  burner  of  a  Primus  lamp  a  powerful  side- 
flame  is  obtained  which  is  almost  as  suitable  for  glass  blowing  as  the  Bunsen  blast 
usually  employed. 

The  ordinary  blast  lamp  used  by  plumbers  is  useful. 


CHAPTER  II 
CULTURE  MEDIA 

WHILE  there  are  certain  advantages  in  sterilizing  the  glass  test-tubes 
prior  to  filling  them  with  media,  yet  this  may  be  dispensed  with — the 
sterilization  after  the  media  has  been  tubed  being  sufficient.  If  a  dress- 
ing sterilizer  is  at  hand,  this  is  preferable  for  sterilizing  such  media  as 
bouillon,  potato,  and  agar  (10  to  15  pounds'  pressure  for  fifteen  min- 
utes) .  Milk  should  be  sterilized  with  the  Arnold,  subjecting  the  media 
to  three  steamings  for  twenty  minutes  on  three  successive  days.  Gela- 
tin may  be  sterilized  in  either  way,  but  preferably  in  the  autoclave  at 
7  pounds'  pressure  for  fifteen  minutes.  As  soon  as  taken  out  of  the 
sterilizer  it  should  be  cooled  as  quickly  as  possible  in  cold  water.  This 
procedure  tends  to  prevent  the  lowering  of  the  melting-point  of  the 
finished  gelatin  and  also  preserves  its  spissitude. 

Blood-serum  is  preferably  solidified  as  slants  in  a  blood-serum  inspissator.  This 
requires  one  to  two  hours.  The  subsequent  sterilization  in  the  autoclave  or  Arnold 
should  not  be  done  immediately  after  making  the  solidified  slants,  but  on  the  subse- 
quent day.  If  done  on  the  same  day,  many  of  the  slants  are  ruined  by  being  dis- 
rupted by  bubbles.  The  preparation  of  blood-serum  slants  or  slants  of  egg  media 
can  be  conveniently  carried  out  in  a  rice  cooker  (double  boiler).  Place  the  tubes  in 
the  inner  compartment  of  the  cooker,  obtaining  the  slant  desired  by  manipulating 
an  empty  test-tube,  or  with  a  towel  or  cotton  batting  on  the  bottom.  Then  cover 
the  tubes  with  another  towel.  The  outer  compartment  should  contain  water  alone 
(not  25%  salt  solution).  The  inner  compartment  should  be  weighted  down  so  that 
it  is  surrounded  by  water — the  light  tubes  not  being  sufficient  to  sink  it.  Allowing 
the  water  in  the  outer  compartment  to  boil  one  or  two  hours  will  inspissate  or  solidify 
the  slants  satisfactorily.  The  sterilization  on  subsequent  days  may  be  carried  out 
in  the  same  apparatus,  although  it  is  more  efficient  if  done  in  an  Arnold  or  an  auto- 
clave. (This  sterilization  in  the  rice  cooker  makes  the  media  too  dry.) 

Rice  Cooker. — In  making  media  a  rice  cooker  is  almost  essential;  at  any  rate,  it  is 
so  if  ease,  expedition,  and  unfailing  success  in  preparation  are  to  be  achieved.  As  it 
is  necessary  to  make  the  contents  of  the  inner  compartment  boil,  the  temperature  of 
the  water  in  the  outer  compartment  must  be  raised.  This  is  done  by  using  a  25% 
solution  of  common  salt  or  a  20%  solution  of  calcium  chloride  in  the  outer  compart- 
ment instead  of  plain  water.  Should  CaCl2  be  carried  over  to  media  in  inner  com- 
partment (as  by  thermometer)  coagulation  of  albumin  and  clearing  of  medium  will 
be  prevented. 

19 


20  CULTURE   MEDIA 

Makers  of  apparatus  for  the  bacteriological  laboratory  now  furnish  a  vessel  for 
making  media  which  has  two  bottoms  with  an  intervening  air  space.  This  hot 
air  layer  prevents  the  scorching  of  the  media  as  is  so  liable  to  occur  when  a  plain 
saucepan  is  used.  Advantages  over  the  rice  cooker  are  that  time  is  saved  in  bringing 
the  media  to  a  boil,  and  also  in  the  maintaining  of  a  brisk  boiling  temperature. 

A  15%  solution  of  salt  raises  the  boiling-point  2^2°C.;  a  20%,  3H°C.,  and  a 
25%,  4K0C.  The  raising  of  the  boiling-point  by  calcium  chloride  is  about  the 
same  for  similar  strength  solutions. 

Although  the  Bacteriological  Committee  of  the  A.  P.  H.  Association  recommends 
special  steps  to  be  taken  in  the  preparation  of  gelatin  and  agar,  yet  for  clinical  pur- 
poses it  will  be  found  satisfactory  to  keep  on  hand  a  stock  of  bouillon,  and  when  it  is 
desired  to  make  agar  or  gelatin  to  simply  prepare  such  media  from  the  stock  bouillon 
in  the  way  to  be  subsequently  given. 


NUTRIENT  BOUILLON 

This  may  be  made  either  from  fresh  meat  or  from  meat  extract. 
Media  from  fresh  meat  are  usually  lighter  in  color  and  possibly  clearer. 
In  the  Philippines,  however,  certain  measures  employed  for  the  preser- 
vation of  the  meat  made  it  very  difficult  to  prepare  clear  bouillon 
from  it,  so  that  meat  extract  was  used  entirely.  There  is  very  little 
difference,  if  any,  in  the  nutritive  power  of  media  made  in  either  way. 

The  chief  objections  to  fresh  meat  as  a  base  are:  i.  It  takes  more  time  and  trouble. 
2.  The  reaction,  due  to  sarcolactic  acid  and  acid  salts,  is  quite  acid,  so  that  it  is 
necessary  to  titrate  and  neutralize  the  excess  of  acidity.  3.  The  reaction  of  the 
finished  media  tends  to  change  unless  the  boiling  at  the  time  of  making  was  very 
prolonged.  4.  It  is  not  infrequent  to  have  a  heavy  precipitate  of  phosphates 
thrown  down  at  the  time  of  sterilization,  thus  making  it  necessary  to  repeat  the 
process  of  nitration  and  sterilization. 

If  fresh  meat  is  used,  take  about  500  grams  (i  pound),  remove  fat  and  cut  it 
up  with  a  sausage  mill  or  purchase  the  meat  already  cut  up  as  for  a  Hamburg  steak. 
It  makes  little  difference  whether  the  amount  be  100  grams  more  or  less.  Place 
the  chopped-up  meat  in  a  receptacle  and  pour  1000  c.c.  of  water  over  it.  Keep  in 
the  ice  chest  over  night  and  the  next  morning  skim  off  with  a  piece  of  absorbent  cot- 
ton the  scum  of  fat;  then  squeeze  out  the  infusion  with  a  strong  muslin  cloth,  mak- 
ing the  amount  up  to  1000  c.c.  This  meat  infusion  contains  all  the  albuminous  mate- 
rial necessary  for  the  clarification  of  the  bouillon.  It  is  convenient  to  designate  this 
meat  base  as  Meat  Infusion  to  distinguish  from  the  base  containing  meat  extract. 

Having  obtained  1000  c.c.  of  this  50%  meat  infusion,  we  dissolve  in  it  i%  of 
Witte's  peptone  and  K%  of  sodium  chloride.  While  there  is  a  sufficiency  of  the  vari- 
ous salts  necessary  for  bacterial  development  in  the  meat  juices,  yet  there  is  not 
enough  to  give  the  best  results  when  bouillon  cultures  of  various  organisms  are  used 
for  agglutination  tests;  and  furthermore,  when  bouillon  is  used  for  blood  cultures, 
disintegration  of  the  red  cells,  with  clouding  of  the  clear  medium,  may  occur  if  there 
t>e  not  sufficient  salt  present  to  prevent  this. 


REACTION  STANDARDIZATION  21 

The  salt  and  the  peptone  are  best  put  in  a  mortar,  and  adding  about  i  ounce 
of  the  meat  infusion  we  make  a  pasty  mass;  then  we  gradually  add  the  remaining 
infusion  until  solution  is  complete.  It  is  sometimes  recommended  to  use  a  tempera- 
ture of  so°C.  to  facilitate  the  solution  of  the  peptone.  This  is  not  necessary,  and 
if  the  temperature  is  not  watched  closely  it  might  go  up  to  65°C.  or  higher  and  we 
should  lose  the  clearing  albuminous  material  from  its  coagulation.  Of  this  rather 
cloudy  solution  take  up  10  c.c.  with  a  pipette  and  let  it  run  out  into  a  porcelain  dish. 
Add  40  c.c.  of  distilled  or  rain  water  and  about  six  drops  of  a  0.5%  phenolphthalein 
solution.  (Phenolphthalein,  0.5;  dilute  alcohol,  100  c.c.)  Bring  the  contents  of 
the  porcelain  dish  to  a  boil  and  continue  boiling  for  one  or  two  minutes  in  order  to 
expel  all  CO2.  Now  from  a  burette  filled  with  decinormal  sodium  hydrate  solution, 
run  in  this  solution  until  we  have  the  development  of  a  faint  but  distinct  pink  in  the 
boiling  diluted  bouillon  which  is  not  dissipated  on  further  boiling. 

It  is  more  satisfactory  to  take  burner  from  beneath  the  porcelain  dish  just  before 
running  in  the  N/io  solution,  again  boiling  so  soon  as  a  pink  color  is  obtained.  Hav- 
ing obtained  the  light  pink  coloration  we  read  off  the  number  of  c.c.  or  fractions  of  a 
c.c.  of  N/io  sodium  hydrate  solution  added  to  produce  the  color.  This  number 
gives  the  acidity  of  the  bouillon  in  percentage  of  N/i  acid  solution. 

Percent  acid  means  that  so  many  c.c.  of  N/i  acid  added  to  100  c.c. 
of  the  medium  at  the  neutral  point  would  give  that  percentage  reaction. 
Thus  ij-^  c.c.  of  N/i  HC1  solution  added  to  100  c.c.  of  medium  at  o, 
would  give  us  ij^%  of  acidity  or  +  1.5.  (Accurately  98^2  c-c-) 

Percent  alkaline  means  so  many  c.c.  of  N/i  sodium  hydrate  solution  added  to 
100  c.c.  of  the  medium  at  the  neutral  point.  Thus  a  %%  alkaline  medium  would  be 
one  whose  alkalinity  would  correspond  to  the  addition  of  ^  c.c.  of  N/i  NaOH  to  100 
c.c.  of  the  medium  at  o.  It  is  written  —0.5. 

If  we  took  100  c.c.  of  the  medium  and  put  it  in  a  beaker  and  then  ran  in  N/i 
NaOH  solution  from  a  burette,  it  will  be  readily  understood  that  if  we  had  to  add 
3^2  c.c.  of  N/i  NaOH  to  obtain  the  pink  color,  it  would  show  that  the  acidity  of  the 
100  c.c.  of  medium,  being  tested,  corresponded  to  3.5  c.c.  of  N/i  acid  solution, 
and  that  its  acidity  was  equal  to  3^%  of  N/i  acid  solution,  or  that  its  reaction  was 
+3-5- 

As  N/i  NaOH  solution  is  too  corrosive  for  general  use  in  a  burette,  and  as  10  c.c. 
of  medium  is  more  convenient  to  work  with  than  100  c.c.,  we  use  a  solution  one- 
tenth  the  strength  of  the  N/i  NaOH  and  we  take  only  one- tenth  of  the  100  c.c.  of 
medium.  In  this  way  it  is  the  same  from  a  standpoint  of  directly  reading  off  our 
percentage  reaction  as  if  we  had  100  c.c.  of  medium  and  used  N/i  NaOH  solution. 
The  A.  P.  H.  Association  recommends  5  c.c.  of  the  medium  and  the  use  of  N/20 
NaOH.  As  the  N/io  NaOH  is  always  at  hand  for  titrating  gastric  juice,  the  N/io 
is  used  instead. 

Should  it  be  found  difficult  to  carry  on  the  titration  while  boiling  the  end  reaction 
may  be  fairly  accurately  determined  in  the  cold.  Deliver  into  a  beaker  from  a 
pipette  10  c.c.  of  the  bouillon  and  make  up  to  50  c.c.  with  distilled  water  and  add  5 
drops  of  0.5%  phenolphthalein  solution.  Then  run  in  N/io  NaOH  from  a  burette 
and  continue  to  add  the  N/io  NaOH  solution  from  the  burette,  drop  by  drop,  until 


22  CULTURE   MEDIA 

the  addition  of  a  drop  fails  to  show  any  intensifying  of  the  purplish-violet  color  at  the 
spot  where  it  came  in  contact  with  the  diluted  bouillon  in  the  beaker.  This  marks 
the  end  reaction.  A  reaction  of  about  +0.7  in  the  cold  gives  a  delicate  pink  with 
phenolphthalein  as  an  indicator.  Titration  in  the  cold  is  not  very  satisfactory  with 
gelatin  and  agar. 

Having  determined  the  percentage  acidity  of  the  10  c.c.  sample  tested,  we  easily 
calculate  the  number  of  c.c  of  N/i  NaOH  solution  required  to  be  added  to  the  1000 
c.c.  of  bouillon  to  obtain  a  reaction  corresponding  to  the  neutral  point  of  phenol- 
phthalein. It  is  more  exact  to  take  the  average  of  two  titrations. 

As  TOO  c.c.  of  medium  would  require  3^  c.c.,  1000  c.c.  would  require  10  times  as 
much,  or  35  c.c.  N/i  NaOH  solution.  Having  measured  out  and  added  35  c.c. 
of  the  N/i  NaOH  solution  to  the  meat  infusion,  containing  salt  and  peptone,  we 
have  a  solution  which  is  exactly  neutral  to  phenolphthalein,  or  o.  It  is  usually  con- 
sidered that  a  reaction  of  about  i%  acid  is  the  optimum  reaction  for  bacterial 
growth.  Hence  we  should  now  add  i%  of  N/i  HC1  solution  to  the  medium.  This 
would  be  accomplished  by  adding  10  c.c.  of  N/i  HC1  solution  to  the  1000  c.c.  of 
neutralized  medium,  and  we  would  have  a  medium  with  a  reaction  of  -f-i.  If  we 
desired  a  reaction  of  i%  alkalinity  we  would  add  an  additional  c.c.  of  N/i  NaOH 
solution  to  every  100  c.c.  of  the  medium  at  o,  or  10  c.c.  for  the  1000  c.c.  of  medium. 
The  reaction  would  then  be  —  i. 

As  a  matter  of  convenience,  we  usually  determine  the  reaction  of  the 
medium,  which  is  always  more  or  less  acid,  and  then  add  enough  N/i 
NaOH  to  reduce  the  acidity  to  the  percentage  we  desire  to  set  the  med- 
ium, instead  of  neutralizing  all  the  acidity  present  and  then,  in  a  second 
operation,  restoring  the  acidity  to  the  point  desired. 

Thus  finding  the  acidity  of  the  medium  to  be  3^%  and  desiring  to  give  it  an 
acidity  of  i%,  we  would  add  only  2%  c.c.  of  N/i  NaOH  to  every  100  c.c.  of  medium, 
or  25  c.c.  for  the  1000  c.c.  of  medium.  The  reaction  would  then  be  found  to 
be  +  i. 

The  neutral  point  of  litmus  is  not  a  sharp  one,  but  it  corresponds  rather  closely 
with  a  reaction  of  +1.5  to  phenolphthalein.  The  recommendations  of  the  A.  P.  H. 
Association  call  for  making  the  titration  with  the  medium  boiling.  If  the  color  of  the 
end  reaction  at  boiling-point  be  obtained,  it  will  be  found  that  when  cool  it  deepens 
until  it  corresponds  to  the  rich  violet-pink  of  the  end  reaction  in  the  cold  or  vice  versa. 

To  summarize: 

Take  Peptone 10  grams 

Sodium  chloride. 5  grams 

50%  meat  infusion 1000  c.c. 

Dissolve  the  peptone  and  sodium  chloride  in  the  meat  infusion  and 
add  enough  N/i  NaOH  to  make  the  reaction  +i. 

Put  the  solution  in  the  inner  compartment  of  a  rice  cooker  and  bring  to  the  boiling- 
point  and  maintain  this  temperature  for  twenty  minutes.  The  calcium  chloride  or 


SUGAR  BOUILLONS  23 

sodium  chloride  in  the  outer  compartment  of  the  rice  cooker  enables  us  to  secure 
a  boiling  temperature  for  the  contents  of  the  inner  compartment.  Do  not  stir  the 
bouillon  that  is  being  heated,  as  the  pultaceous  membranous  mass  of  coagulated 
albumin  makes  filtration  easy.  Filter.  The  filter-paper  in  the  funnel  should  be 
thoroughly  wet  with  water  before  pouring  on  the  bouillon.  This  is  to  prevent 
clogging  of  the  pores  of  the  filter-paper.  Make  up  the  quantity  of  filtrate  to  1000 
c.c.  by  adding  water. 

If  greater  exactness  is  demanded  than  answers  for  ordinary,  clinical  work,  it  is 
advisable  to  again  titrate  and  again  adjust  the  reaction  or  to  simply  record  the  exact 
reaction.  It  is  more  convenient  to  have  a  counterpoise  to  balance  the  inner  com- 
partment and  then  to  add  water  to  the  medium  until  a  kilo  weight,  in  addition  to  the 
weight  balancing  the  container,  is  just  balanced.  Then  titrate,  adjust  the  reaction 
(if  so  desired),  and  filter.  Sterilize  in  the  autoclave  at  i i5°C.  for  fifteen  minutes  or  in 
the  Arnold  on  three  successive  days.  The  use  of  a  balance  is  preferable  in  the 
preparation  of  bouillon,  necessary  in  making  gelatin  and  imperative  in  making 
agar  media. 


BOUILLON  MADE  FROM  LIEBIG'S  MEAT  EXTRACT 

Place  in  a  mortar  3  grams  of  Liebig's  extract,  10  grams  of  peptone  and  5  grams 
of  sodium  chloride.  Dissolve  the  whites  of  one  or  two  eggs  in  1000  c.c.  of  water. 
Then  add  this  egg-white  water,  little  by  little,  to  the  extract,  peptone,  and  salt  in  the 
mortar  until  a  brownish  solution  is  obtained.  Pour  this  into  the  inner  compartment 
of  a  rice  cooker;  apply  heat  to  the  outer  compartment  containing  the  salt  or  calcium 
chloride  solution,  allow  to  come  to  a  boil  and  to  continue  boiling  for  fifteen  to  twenty 
minutes.  Do  not  stir.  Place  inner  compartment  on  the  scales  and  its  counterpoise 
and  a  one-kilo  weight  on  the  other  side.  Add  water  until  the  two  arms  balance. 
Filter  and  sterilize. 

The  reactions  of  media  made  with  Leibig's  meat  extract  rarely  exceeds  +0.75 
(from  -f  0.6  to  +0.9).  Consequently  for  growing  bacteria  it  is  unnecessary  to 
titrate  and  adjust  reactions  unless  precision  is  demanded. 


SUGAR-FREE  BOUILLON 

Inoculate  nutrient  bouillon  in  a  flask  with  the  colon  bacillus.  Allow  to  incubate 
at'37°C.  over  night.  Pour  the  contents  into  a  sauce-pan  and  bring  to  a  boil  to  kill 
the  colon  bacilli.  Put  about  15  grams  of  purified  talc  (Talcum  purificatum,  U.  S.  P.) 
in  a  mortar.  Add  the  dead  colon  culture,  stirring  constantly.  Then  filter  through 
filter-paper.  It  may  be  necessary  to  again  pass  the  filtrate  through  the  same  filter 
until  the  sugar-free  bouillon  is  perfectly  clear. 

For  ordinary  purposes  the  very  small  amount  of  sugar  in  bouillon  made  from 
Liebig's  meat  extract  may  be  neglected  in  determining  gas  production;  so  that  under 
such  conditions  the  various  sugars  could  be  added  directly  to  the  meat-extract  bouil- 
lon. Dunham's  peptone  solution  may  be  used  as  a  substitute  for  sugar-free  bouillon, 
the  sugars  being  added  to  it.  We  prefer  the  serum  water  medium  of  Hiss. 


24  CULTURE   MEDIA 

SUGAR  BOUILLON 

The  sugar  media  ordinarily  used  for  determining  fermentation  or  gas  production 
are  those  of  glucose  and  lactose.  In  special  work  such  carbohydrates  as  saccharose 
and  maltose  are  used.  The  alcohol  mannite  is  used  in  differentiating  strains  of 
dysentery  bacilli. 

To  make,  simply  dissolve  i  or  2  %  of  the  sugar  in  sugar-free  bouillon  or  that  made 
from  meat  extract.  Tube  in  Durham's  or  the  ordinary  fermentation  tubes  and 
sterilize  in  the  autoclave  at  only  about  5  pounds'  pressure  for  fifteen  minutes,  or  in 
the  Arnold.  Ordinary  peptone  solution  is  a  good  substitute  for  sugar-free  bouillon. 

Too  high  a  degree  of  heat  may  turn  the  sugar  bouillon  brownish.  The  nature 
of  the  sugar  itself  may  further  be  affected  by  too  high  a  temperature.  Many  of 
the  carbohydrates  added  to  bouillon  are  liable  to  be  split  up  on  subjection  to  any 
marked  sterilization.  Maltose  is  particularly  unstable.  It  is  best  to  make  about 
20%  solution  of  the  carbohydrates  in  distilled  water  and  sterilize  in  small  flasks  adding 
enough  to  the  sterile  bouillon  to  give  a  i  %  solution.  Inulin  usually  contains  resist- 
ant spores  so  that  its  sterilization  may  need  the  autoclave.  One  of  the  great  diffi- 
culties about  reporting  on  sugar  reactions  is  the  possibility  of  not  working  with  a 
chemically  pure  sugar  as  well  as  with  one  changed  by  too  much  heat.  Inulin  is  a 
polysacchoride  resembling  starch  but  does  not  give  the  iodine  reaction.  It  is 
obtained  from  the  roots  of  chicory  or  dandelion.  For  further  notes  on  carbohydrates 
see  water  analysis. 

BESREDKA'S  EGG  BOUILLON 

ioo  c.c.  broth  without  salt;  80  c.c.  10%  egg-white  solution;  20  c.c.  10%  yolk  of 
egg  solution. 

The  egg-white  solution  is  prepared  by  constant  heating  while  adding  distilled 
water  little  by  little.  It  is  then  filtered  through  absorbent  cotton  and  heated  to 
ioo°C.  Then  filter  through  filter-paper. 

This  opalescent  liquid  is  then  put  into  tubes  or  flasks  and  sterilized  at  ns°C.  for 
twenty  minutes. 

To  ioo  c.c.  of  -10%  emulsion  of  egg  yolk  in  water  you  add  about  i  c.c.  of  N/i 
NaOH  solution.  One  should  add  enough  of  this  caustic  soda  solution  to  clarify  the 
emulsion  slightly;  it  should  still  be  opaque  when  in  a  rather  thick  layer.  Heat  to 
ioo°C.,  then  filter  and  finally  sterilize  at  ii5°C.  for  twenty  minutes. 

The  bouillon  is  a  50  to  75%  meat  infusion  with  peptone. 

This  is  an  excellent  medium  for  growing  pneumococci,  streptococci,  meningococci 
and  gonococci;  even  the  organism  of  pertussis  will  grow  on  this  medium.  Of  course 
such  organisms  as  the  typhoid-colon  group,  cholera,  diphtheria  and  pathogenic 
anaerobes  grow  readily  on  it. 

The  medium  is  particularly  recommended  for  growing  tubercle  bacilli.  For  this 
purpose  the  peptone  in  the  bouillon  is  omitted  as  it  is  found  that  tubercle  bacilli 
grow  better  without  it.  Besredka  found  that  tubercle  bacilli  grew  better  when  he 
omitted  the  addition  of  glycerine  to  his  medium. 

It  will  be  noted  that  for  tubercle  bacilli  the  medium  should  contain  neither  pep- 
tone, salt  nor  glycerine. 

In  the  course  of  two  to  three  weeks  we  have  a  thick  membranous  growth. 


INDOL  PRODUCTION  25 

It  is  stated  that  after  a  growth  of  from  four  to  six  weeks  human  strains  show  a  dry 
scale-like  growth  which  is  easily  detached  from  the  sides  of  the  flask,  while  bovine 
cultures  show  a  sort  of  mucoid  growth  adhering  to  the  walls. 

Egg  Broth.  —  This  has  been  extensively  used  in  the  war  zone  for  growing  anaerobes. 
Emulsify  one  whole  egg  in  300  c.c.  water.  Bring  slowly  to  a  boil  with  frequent 
shaking.  Then  tube  and  sterilize.  The  organism  of  malignant  cedema  grows  par- 
ticularly well  in  it. 

CALCIUM  CARBONATE  BOUILLON 

Where  we  wish  to  cultivate  such  organisms  as  streptococci  and  pneumococci  in 
massive  cultures  we  may  add  small  fragments  of  marble  (calcium  carbonate)  so 
that  any  inimical  excess  of  acid  may  be  neutralized.  North  used  a  glucose  bouillon 
containing  calcium  carbonate  in  the  production  of  massive  cultures  of  B.  bulgaricus. 

GLYCERINE  BOUILLON 

Add  6%  of  glycerine  to  ordinary  bouillon.  It  is  chiefly  used  in  the  cultivation  of 
tubercle  bacilli. 

PEPTONE  SOLUTION  (DUNHAM'S) 


Dissolve  i%  of  Witte's  peptone  and  K%  of  sodium  chloride  in  distilled  water. 
Filter,  tube,  and  sterilize.  Peptone  soluTioiTmay  be  used  as  a  base  for  sugar  media 
instead  of  bouillon.  If  is  the«medium  used  in  testing  for  indol  production.  This 
test  is  made  by  adding  from  6  to  8  drops  of  concentrated  H2SO4  to  a  twenty-four-  to 
forty-eight-hour-old  peptone  culture  of  the  organism  to  be  tested.  If  the  organism 
produces  both  indol  and  a  nitroso  body,  we  obtain  a  violet-pink  coloration,  "cholera 
red."  If  no  pink  color  is  produced  on  the  addition  of  the  sulphuric  acid,  add  about 
i  c.c.  of  an  exceedingly  dilute  solution  (i  :  10,000)  of  sodium  nitrite. 

Cholera  Red.  —  It  is  very  important  in  determining  the  "cholera  red"  reaction 
to  know  that  the  peptone  used  will  give  the  reaction  as  it  is  not  given  by  true 
cholera  strains  with  certain  samples  of  peptone. 

For  the  Voges-Proskauer  Reaction.  —  Fill  fermentation  tubes  with  a  2%  glucose 
Dunham's  peptone  solution  and  sterilize.  After  inoculation  with  the  organism  to  be 
tested  incubate  for  three  days.  Then  add  2  to  3  c.c.  of  strong  caustic  potash 
solution.  The  development  of  a  pink  color  on  exposure  to  the  air  is  a  positive  reac- 
tion (the  color  of  a  weak  eosin  solution). 


Hiss'  SERUM  WATER  MEDIUM 

Take  one  part  of  clear  beef  €erum  and  add  to  it  about  three  times  its  bulk  of 
water.  Heat,  the  mixture  in  the  ?Ernold  for  fifteen  minutes  to  destroy  any  diastatic 
ferment  which  might  be  present.  Color  to  a  deep  transparent  blue  with  litmus 
solution  and  then  add  i  %  of  any  of  the  various  sugars  used  in  fermentation  tests. 
Sterilize  in  the  Arnold  by  the  fractional  method. 


26  CULTURE   MEDIA 

NUTRIENT  AGAR 

In  making  agar  medium  it  is  preferable  to  use  powdered  agar,  as  this  goes  into 
solution  more  readily  than  the  shredded  agar.  The  reaction  of  agar  is  slightly  alka- 
line, so  that  if  1 3^  to  2%  of  agar  is  added  to  nutrient  bouillon  having  a  reaction 
of  +i  the  finished  product  will  be  found  to  be  about  +0.8. 

To  make:  Weigh  out  15  to  20  grams  of  powdered  agar  and  place  in  a  mortar. 
Make  a  paste  by  adding  nutrient  bouillon,  little  by  little,  and  when  a  smooth  even 
mixture  is  made,  pour  it  into  the  inner  compartment  of  a  rice  cooker  and  add  the 
remainder  of  the  1000  c.c.  of  bouillon.  The  use  of  the  balance  is  preferable. 

The  outer  compartment  of  the  rice  cooker  should  contain  the  25%  salt  solution. 
Bring  to  boll,  and  the  agar  will  be  found  to  have  entirely  gone  into  solution  after 
five  to  ten  minutes  of  boiling. 

Then,  using  a  funnel  which  has  been  heated  in  boiling  water  and  which  contains 
a  small  pledget  of  absorbent  cotton,  or  the  cotton  between  two  layers  of  gauze,  we 
filter  the  agar,  tube  it,  and  sterilize  it  in  the  autoclave  or  Arnold.  One  and  one-half 
percent  agar  can  be  readily  filtered  through  filter-paper  and  gives  a  clearer  medium. 

Some  prefer  to  place  the  filter  stand,  funnel  with  gauze  cotton  filter  and  flask  in 
an  Arnold  sterilizer,  for  twenty  minutes,  so  that  when  taken  out  the  funnel  will  not 
only  be  hot  but  the  filter,  being  moist,  will  allow  of  more  rapid  filtration. 

By  taking  of  meat  extract  3  grams,  peptone  10  grams,  salt  5  grams, 
powdered  agar  15  grams,  the  white  of  one  egg  and  1000  c.c.  of  water, 
making  at  first  a  paste  of  all  the  ingredients  in  a  mortar,  then  gradually 
adding  the  remainder  of  the  1000  c.c.  of  water,*putting  in  the  rice  cooker, 
bringing  to  a  boil  without  stirring,  allowing  to  boil  fifteen  minutes  and 
then  filtering  through  absorbent  cotton  placed  between  two  layers  of 
gauze  in  a  hot  funnel,  we  obtain  a  satisfactory  medium,  the  reaction  of 
which  will  be  from  +0.7  to  +0.9.  It  is  very  important  not  to  interfere 
with  the  pultaceous  coagulum  which  forms  on  the  surface  of  the  boiling 
agar. 

Where  very  exact  adjustment  of  the  reaction  of  the  finished  product  is  desirable 
the  method  of  preparation  of  the  Committee  on  Water  Analysis  of  the  American 
Public  Health  Association  is  to  be  preferred. 

Dissolve  15  grams  of  agar  in  500  c.c.  of  water  in  the  inner  compartment  of  the 
rice  cooker  previously  described.  After  the  agar  is  in  solution  (after  ten  to  fifteen 
minutes  boiling)  remove  the  inner  compartment,  containing  the  3  %  agar  solution,  and 
allow  it  to  cool  to  about  SS°C.  Mix  in  the  mortar,  as  described  in  the  directions  for 
making  nutrient  bouillon  from  Liebig's  extract,  3  grams  of  Liebig's  extract,  10  grams 
of  peptone  and  5  grams  of  sodium  chloride  in  500  c.c.  of  water  containing  the  whites  of 
one  or  two  eggs.  Heat  this  mixture  to  50  to  S5°C^kid  pour  it  into  the  agar  solution, 
in  the  inner  compartment,  which  has  been  cooled  to  about  55°C.  Now  titrate  this 
mixture  containing  500  c.c.  of  double  strength  agar  and  500  c.c.  of  double  strength 
peptone,  meat  extract  and  salt  solution.  The  resulting  1000  c.c.  gives  i^%  agar 
and  i%  peptone  solution.  Having  adjusted  the  reaction  by  the  addition  of  the 


GONOCOCCUS  STARCH  MEDIUM  27 

necessary  amount  of  N/i  acid  or  alkali,  we  place  the  inner  compartment  in  the  outer 
one  of  the  rice  cooker,  bring  to  a  boil  and  filter  through  filter-paper  which  has  been 
wetted  with  boiling  water.  The  filtration  can  be  carried  out  in  the  autoclave  or  in  an 
Arnold  sterilizer.  Of  course  the  ordinary  filtering  through  gauze  and  cotton  will 
answer  where  clearer  media  is  not  an  object. 


GLUCOSE  AGAR 

Add  the  agar  to  i  or  2%  glucose  bouillon  and  pro  eed  as  for  ordinary  agar.  If 
preferred,  the  glucose  agar  can  be  made  by  rubbing  up  meat  extract  3  grams,  peptone 
10  grams,  salt  5  grams,  glucose  10  grams  and  15  grams  of  agar  in  1000  c.c.  of  water 
containing  the  white  of  egg  (one  to  two  eggs),  then  boiling  in  the  rice  cooker  and 
filtering. 

GLYCERINE  AGAR 

Add  the  agar  to  6%  glycerine  bouillon  instead  of  nutrient  bouillon,  or  the  gly- 
cerine may  be  added  to  nutrient  agar  which  has  been  melted.  Glycerine  agar  with  a 
reaction  of  o  makes  an  excellent  base  for  blood  and  serum  media  for  use  in  culturing 
delicate  pathogens. 

GLYCERINE  AGAR  EGG  MEDIUM 

Take  the  white  and  the  yolk  of  one  egg  and  mix  thoroughly  in  a  vessel  kept 
between  45°  and  55°C.  with  an  equal  amount  of  glycerine  agar.  Tube  the  medium, 
inspissate  in  a  rice  cooker  as  for  serum  tubes,  and  sterilize  as  for  blood-serum  tubes. 

This  makes  an  excellent  medium  for  growing  tubercle  bacilli.  As  egg  medium  has 
a  tendency  to  be  dry,  it  is  well  to  add  i  c.c.  of  glycerine  bouillon  to  each  slant  before 
autoclaving. 

VEDDER'S  STARCH  MEDIUM 

Macerate  500  grams  of  beef  in  1500  c.c.  of  water  in  ice  box  over  night.  In  the 
morning  filter  into  stew  pan  through  doubled  gauze.  Bring  fluid  slowly  to  a  boil  and 
add  agar  iH%-  Boil  for  from  twenty  to  thirty  minutes. 

Titrate  and  correct  reaction  to  0.2  to  0.7  acid.  Check  reaction  to  show  faint  blue 
on  red  litmus. 

Let  cool  to  about  6o°C.  and  add  two  whole  eggs,  well  beaten,  to  clarify.  Bring 
very  quickly  to  a  boil,  then  turn  down  flame  and  allow  to  simmer  until  a  complete 
coagulum  forms. 

Filter  through  gauze  and  cotton  and  add  i  %  starch. 

Let  stand  in  Arnold  for  about  forty-five  minutes,  shaking  about  three  times  during 
this  period,  to  distribute  the  starch. 

Tube  or  flask,  and  sterilize  in  autoclave  for  fifteen  minutes  at  10  pounds.  There 
must  be  plenty  of  water  of  condensation  and  the  agar  must  not  exceed  i>£%. 

This  is  especially  recommended  for  culturing  gonococci.  English  workers  have 
used  it  in  isolating  meningococci  from  carriers.  For  this  purpose  the  starch  agar 
has  i%  of  glucose  added  to  it  and  colored  with  litmus  solution.  The  Meningococcus 
acidifies  the  glucose  while  the  M.  Catarrhalis  does  not. 


28  CULTURE   MEDIA 

NUTRIENT  GELATIN 

Place  in  a  mortar  3  grams  of  Liebig's  extract,  10  grams  of  peptone  and  5  grams 
of  sodium  chloride.  Dissolve  the  whites  of  one  or  two  eggs  in  1000  c.c.  of  water. 
Then  add  this  egg-white  water,  little  by  little,  to  the  meat  extract,  peptone  and  salt, 
in  the  mortar,  until  a  brownish  solution  is  obtained.  Pour  this  into  the  inner  com- 
partment of  the  rice  cooker  and  bring  the  temperature  up  to  45°C.  (This  prelim- 
inary elevation  of  temperature  is  better  carried  out  in  some  heated  water  in  a  pan, 
as  the  heating  by  means  of  the  salt  solution  in  the  outer  compartment  of  the  rice 
cooker  is  difficult  to  control,  so  that  a  temperature  approximating  7o°C.  might  be 
obtained  and  the  albumin  of  the  white  of  egg  coagulated.  The  temperature  in 
the  outer  compartment  might  be  approaching  boiling  before  the  contents  of  the  inner 
compartment  would  show  45°C.)  Now  take  about  120  grams  of  "gold  label"  or 
other  good  quality  gelatin  (12%)  and  crush  it  down  in  the  meat  extract  egg- water 
solution  in  the  inner  compartment  of  the  rice  cooker. 

The  gelatin  quickly  goes  into  solution  at  45°C.  Gelatin  being  quite  acid  it  will 
probably  be  found  upon  titration  that  the  reaction  is  about  +4%.  '  N/i  NaOH 
solution  is  added  to  bring  the  reaction  to  about  +1%  or  3  c.c.  of  N/i  NaOH  for 
each  100  c.c.,  provided  the  reaction  were  exactly  +4%-  The  procedure  is  the  same 
as  for  bouillon.  The  color  reaction  is  not  quite  as  distinct  with  gelatin  as  with 
bouillon. 

Having  neutralized  and  allowed  to  boil  for  fifteen  minutes,  we  filter  through  filter- 
paper  in  a  hot  funnel.  As  it  is  very  important  that  gelatin  should  be  perfectly 
clear,  it  is  better  to  filter  through  filter-paper  than  through  cotton.  The  filter-paper 
should  be  very  thoroughly  wetted  with  very  hot  water  before  filtering  gelatin  or  agar. 

Tube  the  medium  and  sterilize,  either  in  the  Arnold  on  three  successive  days  or  in 
the  autoclave  at  8-10  pounds'  pressure  for  ten  minutes.  The  tubes  should  be 
cooled  as  quickly  as  possible  in  cold  water  after  taking  out  of  the  sterilizer. 

AGAR  GELATIN  MEDIUM  (NORTH) 

Lean  chopped  beef  or  veal 500  grams. 

Agar 10  grams. 

Gelatin,  Gold  label 20  grams. 

Peptone,  Witte's, 20  grams. 

Sodium  chloride 5  grams. 

Distilled  water,  q.s •.    1000  c.c. 

Extract  the  chopped  beef  with  500  c.c.  distilled  water  for  eighteen  hours,  strain 
through  muslin  and  combine  the  ingredients  in  the  usual  way.  Adjust  the  reaction 
to  the  neutral  point,  using  phenolphthalein  as  indicator. 

North  states  that  this  medium  is  excellent  for  streptococci,  pneumococci  and 
diphtheria  bacilli  because  it  is  soft,  moist,  and  can  be  used  at  37°C. 

It  is  claimed  to  be  of  special  value  for  carrying  stock  cultures.  It  is  useful  as  a 
plating  medium  for  milk. 

LITMUS  MILK 

Milk  for  media  should  be  as  fresh  as  possible.  It  should  then  be  put  in  a  1000- 
c.c.  Erlenmeyer  flask,  sterilized  for  fifteen  minutes  in  the  Arnold,  and  set  over  night 


AZOLITMIN  2Q 

in  the  refrigerator.  The  next  morning  the  milk  beneath  the  cream  should  be 
siphoned  off.  The  short  arm  of  the  siphon  should  not  reach  the  bottom  of  the  flask 
so  as  to  avoid  the  sediment.  Add  sufficient  litmus  solution  to  this  milk  to  give  a 
decided  lilac  tinge;  tube  and  sterilize  in  the  Arnold  on  three  successive  days. 

Litmus  milk  which  apparently  is  as  satisfactory  as  the  above  as  regards  nutritive 
quality  and  cultural  characteristics  can  be  made  from  certain  canned  milks  which 
have  not  been  condensed  or  sweetened  and  which  do  not  contain  chemical  pre- 
servatives. The  "Natura"  brand  of  milk  is  the  one  I  have  experimented  with. 

Litmus  Solution. — A  simple  solution  may  be  made  by  digesting  the  powdered 
cubes  repeatedly  with  hot  water,  mixing  the  extracts,  and,  after  allowing  them  to 
stand  all  night,  decanting  the  solution  from  the  inert  sediment  into  a  clean  bottle. 

In  litmus  solution  so  made,  however,  a  red  dye  is  also  present  while  calcium  and 
other  salts  are  dissolved  out.  For  bacteriological  purposes  a  pure  solution  of  the 
blue  dye  should  be  used.  This  is  called  "azolitmin."  It  is  freely  soluble  in  water 
but  insoluble  in  alcohol. 

It  can  be  conveniently  prepared  as  follows:  Weigh  out  2  ounces  of  powdered 
litmus;  digest  repeatedly  with  fresh  quantities  of  hot  water  until  all  the  coloring 
matter  is  dissolved  out;  allow  to  settle,  and  decant  off  the  fluid  from  the  insoluble 
powder.  Add  together  the  extracts,  which  should  measure  about  a  liter.  Evapo- 
rate down  the  solution  to  a  moderate  bulk,  then  add  a  slight  excess  of  acetic  acid, 
so  as  to  convert  all  carbonates  present  into  acetates.  Continue  the  evaporation, 
the  later  stages  over  a  water  bath,  until  the  solution  becomes  pasty.  Add  200  c.c. 
of  alcohol,  and  mix  thoroughly.  The  alcohol  precipitates  the  blue  coloring  matter, 
while  a  red  coloring  matter,  together  with  the  alkaline  acetate  present,  remains  in 
solution.  Transfer  to  a  filter.  Wash  out  the  dish  with  alcohol  and  add  this  to 
the  filter.  Wash  the  precipitate  on  the  filter  with  alcohol.  Dissolve  the  pure  color- 
ing matter  remaining  on  the  filter  in  warm  distilled  water  and  dilute  to  500  c.c. 
Azolitmin  solution  prepared  in  this  way  is  more  sensitive  than  ordinary  litmus 
solution. 

Azolitmin  in  powder  can  be  purchased  from  dealers  in  chemicals. 


POTATO  SLANTS 

Take  Irish  potatoes  and  scrub  thoroughly  with  a  stiff  brush.  Then  pare  off 
generously  all  the  outer  portion.  From  the  white  interior  cut  out  cylinders  with  a 
cork  borer.  These  cylinders  should  be  of  Y^  to  %  of  an  inch  in  diameter.  Divide 
a  cylinder  by  a  diagonal  cut.  This  gives  a  plug  with  a  flat  base,  the  other  extremity 
being  a  slant.  These  potato  plugs  should  be  left  in  running  water  over  night  or 
washed  with  frequent  changes  of  water.  This  prevents  the  blackening  of  the  plug. 
Into  a  i-inch  test-tube  drop  a  pledget  of  absorbent  cotton  well  moistened  with  water. 
Then  drop  in  the  potato  plug,  base  downward.  Sterilize  in  the  autoclave  at  15 
pounds  for  fifteen  to  twenty  minutes,  to  insure  sterility. 

For  glycerine  potato,  soak  the  plugs  in  6%  glycerine  solution  for  about  one 
hour.  Then  drop  a  pledget  of  absorbent  cotton  moistened  with  the  same  glycerine 
solution  into  the  test-tubes  and  follow  it  with  the  potato  plug.  Sterilize  in  the 
autoclave. 


30  CULTURE   MEDIA 

BLOOD-SERUM 

The  blood  of  cattle  should  be  collected  in  large  pans  or  pails  at  the  abattoir. 
This  vessel  of  blood  should  then  be  kept  in  the  cold-storage  room  and  the  next  morn- 
ing the  more  or  less  clear  serum  will  have  been  squeezed  out  from  the  clot.  Collect 
this  serum  and  keep  in  the  ice  chest  for  future  use.  If  to  be  kept  for  a  long  time,  it 
is  advisable  to  add  about  2%  of  chloroform  to  the  serum  in  tightly  corked  flasks. 
This  will  not  only  keep  the  serum,  but  will  eventually  sterilize  it. 

To  make  Loffler's  serum,  take  i  part  of  glucose  bouillon  and  3  parts  of  blood- 
serum.     Mix,  tube,  and  coagulate  the  albumin  in  the  inspissator  or  rice  cooker, 
giving  the  tubes  a  proper  slant  before  heating.     Sterilize  the  following  day  in  tl 
autoclave  as  previously  directed  (7  pounds)  or  in  the  Arnold  on  three  successive  da} 

A  SUBSTITUTE  FOR  ORDINARY  BLOOD-SERUM 

Add  from  10  to  15  c.c.  of  i%  glucose  bouillon  to  the  white  and  yolk  of  one  egg, 
make  a  smooth  mixture  in  a  mortar  and  tube.  Inspissate  and  sterilize  as  for  ordi- 
nary serum  slants.  The  morphology  of  the  diphtheria  bacilli  and  the  luxuriance  of 
growth  is  similar  to  that  of  cultures  on  Loffler's  serum. 

When  this  medium  is  to  be  used  for  culturing  tubercle  bacilli  add  about  i  c.c. 
of  glycerine  bouillon  to  each  tube  before  final  sterilization  in  the  autoclave.  The 
cotton  plugs  should  be  paraffined  to  prevent  drying  of  the  slants  in  the  incubator. 
This  medium  seems  to  answer  as  a  substitute  for  Dorsett's  egg  medium.  (While 
glycerine  bouillon  favors  growth  of  human  tuberculosis,  it  is  not  so  satisfactory  fc 
bovine  tuberculosis  as  plain  glucose  bouillon.)  This  is  better  than  the  various  whit( 
of  egg  substitutes  usually  recommended.  (Pouring  a  little  alcohol  in  the  mort< 
and  moistening  the  sides  by  tilting,  then  burning  off  the  alcohol,  in  a  measure 
sterilizes  the  mortar.  If  the  egg  is  cracked  open  with  a  sterile  knife,  a  medium  can 
be  prepared  which  will  be  sterile  as  the  result  of  the  two-hour  inspissation  in  the  rice 
cooker.)  By  covering  the  tube  with  a  rubber  cap  or  preferably,  by  heating  the 
plugged  end  of  the  test-tube,  quickly  withdrawing  the  cotton  plug  and  dipping  the 
part  of  the  plug  which  enters  the  tube  into  hot  melted  paraffin,  then  quickly  reintro- 
ducing  the  plug,  the  contents  of  the  tube  will  be  prevented  from  drying  out.  This 
procedure  is  essential  for  growing  tubercle  bacilli. 

Egg  media  are  excellent  for  culturing  anaerobes.  One  can  add  about  5  drops  of 
i%  neutral  red  aqueous  solution  to  each  egg,  inspissating  as  above.  The  reddish 
color  appears  in  colonies  producing  acid  and  is  of  value  in  anaerobic  work  to  make 
such  colonies  more  distinct. 

DORSETT'S  EGG  MEDIUM 

This  is  prepared  by  breaking  whole  eggs  into  a  sterile  flask,  mixing  thoroughly  then 
adding  25  c.c.  water  to  every  4  eggs,  straining  through  a  sterile  cloth  and  tubing  10 
c.c.  quantities.  These  tubes  are  slanted  in  an  inspissator  and  kept  at  73°C.  for 
four  or  five  hours  on  two  successive  days.  On  the  third  day  a  temperature  of  76°C.  is 
applied.  Before  inoculating  add  3  or  4  drops  of  sterile  water  to  each  tube.  The 
tuberculous  material  should  be  rubbed  into  the  surface  well  and  the  plugs  paraffined. 


BLOOD  AGAR  31 

HYDROCELE,  SERUM,  ASCITIC  AND  MILK  AGAR 

To  tubes  of  melted  agar  at  5o°C.,  add  from  i  to  3  c.c.  of  hydrocele  or  ascitic  fluid, 
observing  aseptic  precautions.  Allow  the  agar  to  solidify  as  a  slant,  or  as  a  poured 
plate. 

For  milk  agar  we  add  2  or  3  c.c.  of  plain  or  litmus  milk  to  a  tube  of  melted  agar. 
This  makes  an  excellent  plating  medium  for  B.  bulgaricus.  When  poured  into  a 
plate  it  is  opaque  but  the  colonies  stand  out  well. 

For  obtaining  sterile  serum  we  generally  use  the  apparatus  described  under 
Blood  Agar  and  as  a  rule  take  the  blood  from  vein  of  arm  in  man  or  vein  of  neck  of 
sheep  used  for  Wassermann  work.  The  sterile  serum  separates  from  the  clot  and 
can  be  pipetted  off  with  a  sterile  pipette  and  added  to  the  melted  2  to  3%  agar  as 
above.  A  method  recommended  by  Fildes  is  to  take  blood  at  the  slaughter  house 
in  sterile  vessels,  allow  to  clot  and  remove  serum.  Five  c.c.  of  ether  is  added  to 
each  100  c.c.  in  a  glass-stoppered  bottle  of  which  the  stopper  is  fixed  in  and  the  mix- 
ture heated  for  one  hour  in  a  water-bath  at  45°C.  after  which  it  is  placed  in  the 
incubator  at  37°C.  for  several  days,  by  which  time  the  serum  is  sterile.  Before  using 
the  ether  is  driven  off  at  45°C.  This  is  better  than  the  old  method  of  sterilizing 
with  2%  chloroform. 

BLOOD  AGAR 

For  obtaining  blood  to  make  blood  agar  we  use  the  apparatus  described  under 
blood  culturing  and  shown  in  Fig.  8.  We  take  human  blood  from  the  arm  vein, 
sheep  blood  from  the  neck  vein,  or  rabbit  blood  from  the  heart.  In  the  Erlenmeyer 
flask  of  100  c.c.  capacity  is  placed  5  c.c.  of  sterile  10%  sodium  citrate  solution  pro- 
vided we  want  to  take  50  c.c.  of  blood  from  sheep  or  man.  The  final  mixture  to 
prevent  coagulation  should  contain  about  i%  sodium  citrate.  As  a  rule  we  only 
take  about  25  c.c.  from  man  or  rabbit  so  2^  c.c.  of  10%  citrate  would. suffice.  The 
perforated  rubber  stopper  is  removed  and  replaced  with  the  sterile  cotton  plug  of 
the  flask  and  the  fluid  mixture  pipetted  off  and  added  to  the  melted  agar. 

Mixture  is  facilitated  by  rotating  the  tube  rapidly  between  the  hands.  The 
medium  may  be  slanted  or  poured  into  plates.  As  this  medium  is  satisfactory 
for  the  growth  of  haemoglobinophilic  organisms-,'  as  well  as  for  others,  we  use  it  as  a 
routine  plating  medium,  the  others  being  nutrient  agar  and  Endo. 

BLOOD-STREAKED  AGAR 

Sterilize  the  lobe  of  the  ear  and  puncture  with  a  sterile  needle.  Collect  the  ex- 
uding blood  on  a  large  platinum  loop  and  smear  it  over  the  surface  of  an  agar  slant. 
It  is  advisable  to  incubate  over  night  as  a  test  for  sterility.  Plates  or  slants  of  glycer- 
ine agar  of  neutral  reaction  smeared  with  blood  give  the  best  results  when  such 
delicate  pathogens  as  pneumococci,  streptococci,  gonococci  or  meningococci  are  to 
be  cultured. 

BILE  MEDIA 

Secure  ox  bile  from  the  abattoir  or  human  bile  from  cases  of  gall-bladder  drainage 
in  hospitals.  Put  about  10  c.c.  in  each  tube  and  sterilize.  Some  prefer  to  add 


32  CULTURE   MEDIA 

i%  of  peptone.     Conradi's  medium  is  ox  bile  containing  10%  of  glycerine  and  2% 
of  peptone.     This  is  the  medium  for  blood  cultures  in  typhoid,  etc. 

The  bile  lactose  medium  now  used  in  water  analysis  is  made  by  adding  i%  of 
lactose  to  ox  bile  and  tubing  in  fermentation  tubes.  As  a  substitute  for  fresh  bile 
one  may  use  a  15  to  20%  solution  of  a  good  quality  of  inspissated  ox  gall  (Pel  Bovis 
Purincatum).  A  liver  bouillon  made  by  using  500  grams  of  finely  divided  beef  liver 
in  1000  c.c.  of  water  with  i%  peptone,  and  prepared  as  for  meat  infusion  broth,  is  a 
good  substitute  for  bile. 

RECTOR'S  BILE  LACTOSE  NEUTRAL  RED  MEDIUM 

This  is  recommended  in  the  isolation  of  the  colon  bacillus  as  superior  to  lactose 
litmus  agar.  It  consists  of  10%  of  dried  ox  bile,  i%  of  peptone,  and  i>£%  agar. 
After  the  medium  is  filtered  and  tubed  we  add  i%  of  lactose  and  i%  of  a  i-ioo 
neutral  red  solution.  Colon  colonies  have  a  distinct  purplish  red  zone.  Further- 
more the  bile  inhibits  the  growth  of  many  organisms  which  give  pink  colonies  on 
lactose  litmus  agar.  MacConkeys'  bile  salt  medium  contains  %%  oi  sodium 
taurocholate  and  is  colored  with  neutral  red. 


THALMAN'S  MEDIUM  FOR  THE  GONOCOCCUS 

Five  hundred  grams  of  lean,  finely  minced  beef  are  placed  in  1000  c.c.  of  distilled 
water  and  allowed  to  stand  over  night  in  an  ice  box.  It  is  then  filtered  and  the  fil- 
trate made  up  to  1000  c.c.  with  distilled  water.  To  100  c.c.  of  the  beef  juice  add 
iM  grams  of  agar,  and  boil  for  15  minutes.  Then  add  2  grams  of  glucose,  and  bring 
the  reaction  to  plus  0.6  by  addition  of  N/i  NaOH.  Tube,  sterilize,  slant,  and  in- 
cubate over  night.  No  peptone  or  salt  is  required.  We  now  use  Vedder's  starch 
agar. 

PETROFF'S  TUBERCLE  BACILLUS  MEDIUM 

This  is  a  remarkably  valuable  medium  for  isolating  tubercle  bacilli  from  sputum  or 
pus  directly.  Fresh  sputum  shouW  be  used  and  for  destruction  of  contaminating 
organisms  it  should  be  shaken  up  with  an  equal  amount  of  3%  NaOH  solution  and 
left  in  the  incubator  for  one  or  two  hours.  Neutralize  with  N/i  HC1,  using  litmus 
paper,  and  centrifugalize.  Take  up  sediment  and  smear  out  on  slants  of  the 
following  medium. 

Treat  500  grams  of  chopped  up  meat  with  500  c.c.  of  15%  glycerine  solution.  Keep 
in  ice  chest  twenty-four  hours  and  filter  through  gauze.  Sterilize  the  shells  of  eggs 
by  immersion  in  70%  alcohol  for  ten  minutes  or  by  dipping  them  in  boiling  water 
for  five  seconds  or  so.  Mix  white  and  yolk  of  these  eggs  in  a  sterile  mortar  and  add 
an  equal  volume  of  the  glycerine  meat  infusion  which  should  have  added  to  it  before 
mixing  i  c.c.  of  i%  alcoholic  solution  of  gentian  violet  to  each  100  c.c.  of  the 
glycerine  meat  infusion. 

Should  one  be  culturing  bovine  strains  the  glycerine  should  be  omitted  from  the 
meat  infusion  but  the  gentian  violet  (1-10,000)  added.  Put  3  to  4  c.c.  of  this  me- 
dium in  test-tubes  and  inspissate  as  slants  at  85°C.  until  the  medium  is  solidified. 


ENDO'S   MEDIUM  33 

Subject  these  slants  to  temperature  of  75°C.  on  the  second  and  third  days  for  one 
hour. 

PLATING  MEDIA  FOR  FAECES  WORK 

The  media  of  Endo,  Conradi-Drigalski  and  the  lactose  litmus  agar  medium  are 
probably  the  most  satisfactory  of  the  numerous  ones  that  have  been  proposed  for 
plating  out  faeces.  A  convenient  way  of  preparing  any  one  or  all  of  these,  and  which 
apparently  gives  media  equal  to  that  prepared  according  to  the  original  formulae, 
is  as  follows: 

Liebig's  extract 5  grams. 

Salt 5  grams. 

Pepton 10  grams. 

Agar 30  grams. 

Water  to  make 1000  c.c. 

Prepare  as  for  ordinary  nutrient  agar,  with  the  difference  that  the  reaction  should 
be  brought  down  to  o.  Some  prefer  a  reaction  of  +0.2. 

A  stiff  agar  (3%)  is  employed  to  check  the  diffusion  of  acid  beyond  the  colony. 


FOR  ENDO'S  MEDIUM 

Keep  this  agar  base  in  100  c.c.  quantities  in  Erlenmeyer  flasks  instead  of  test- 
tubes.  (If  more  convenient  smaller  quantities  may  be  put  in  the  flask.)  When 
needed  for  plating,  melt  a  flask  of  this  agar,  and  while  liquid  add  to  the  100  c.c. 
6  drops  of  a  saturated  alcoholic  solution  of  basic  fuchsin,  and  then  about  20  drops 
of  a  freshly  prepared  10%  solution  of  sodium  sulphite.  The  sulphite  solution  de- 
colorizes the  intense  red  of  the  fuchsin  to  a  light  rose  pink.  This  color  fades  to  a 
light  flesh  or  pale  salmon  color  when  cold.  Now  add  5  c.c.  of  a  freshly  prepared 
hot  aqueous  20%  solution  of  chemically  pure  lactose.  If  only  occasionally  using 
such  media,  tube  in  "20  c.c.  quantities  and  add  i  drop  of  the  basic  fuchsin  and  4 
drops  of  the  sodium  sulphite  solution  and  i  c.c.  of  the  hot  freshly  prepared  lactose 
solution  to  a  tube  of  the  melted  agar  base  just  before  pouring  the  plate.  This  me- 
dium contains  i  %  of  lactose.  Kendall  prepares  an  Endo  medium  which  only  con- 
tains iH%  of  agar  and  with  a  reaction  just  alkaline  to  litmus  (about  plus  1.2%). 

Colon  bacilli  show  on  this  medium  as  vermilion  colonies,  which  in  about  forty- 
eight  hours  have  a  metallic  scum  on  them.  Typhoid  and  dysentery  colonies  are 
grayish.  Streptococci  a  deep  red. 

FOR  LACTOSE  LITMUS  AGAR 

Color  the  100  c.c.  of  agar  base  with  litmus  solution  to  a  lilac  color.  Then  add 
5  c.c.  of  the  hot  freshly  prepared  20%  lactose  solution  in  distilled  water.  This 
may  be  tubed,  putting  10  c.c.  in  each  test-tube,  or  put  in  quantities  of  50  or 
100  c.c.  in  small  Erlenmeyer  flasks.  It  is  then  sterilized  in  the  autoclave  (10 
pounds  for  fifteen  minutes)  or  in  the  Arnold. 
3 


34  CULTURE   MEDIA 

FOR  CONRADI-DRIGALSKI  MEDIUM 

To  ioo  c.c.  of  lactose  litmus  agar  add  i  c.c.  of  a  solution  of  crystal  violet  (crystal 
violet  o.i  gram,  distilled  water  ioo  c.c.).  The  medium  is  then  ready  to  put  into 
plates.  Colon  colonies  are  pink.  Typhoid  and  dysentery  colonies,  a  bluish-gray. 

CONRADI'S  BRILLIANT  GREEN  MEDIUM 

Take  of  Liebig's  extract  20  grams  (2%),  peptone  10  grams  (i%),  agar  30  grams 
(3%)  and  water  to  1000  c.c.  This  amount  of  meat  extract  should  give  about  the 
proper  acidity,  +3.  If  not,  the  reaction  should  be  adjusted  to  that  point.  Filter 
through  cotton,  tube  150  c.c.  amounts  into  250  c.c.  Erlenmeyer  flasks  and  sterilize. 

Then  add  i  c.c.  of  a  i  to  1000  aqueous  solution  of  brilliant  green  (Hochst)  and 
i  c.c.  of  a  i%  solution  of  picric  acid  to  the  flasks  containing  150  c.c.  of  the  melted 
agar.  Sterilization  after  adding  the  dyes  precipitates  them  and  is  unnecessary. 
Pour  the  finished  medium  into  large  Petri  dishes  and  inoculate  the  surface  with  the 
faeces. 

Brilliant  green  does  not  interfere  with  agglutination  as  does  malachite  green. 

This  medium  is  considered  by  some  authorities  the  one  of  choice  in  isolating 
typhoid  bacilli  from  fasces  and  urine. 

The  surface  of  the  poured  plates  of  Endo,  Conradi-Drigalski,  and  the  brilliant 
green  media  should  be  dried  in  the  incubator  before  smearing  with  the  fasces.  For 
routine  work  I  prefer  Endo's  medium  followed  by  Russell's  double  sugar  agar. 

SELECTIVE  MEDIA  FOR  CHOLERA 

Dieudonne's  medium  rests  on  the  ability  of  cholera  to  grow  when  alkali  is  present 
in  such  amounts  as  to  inhibit  the  growth  of  other  faecal  bacteria. 

Take  equal  parts  of  defibrinated  blood  obtained  at  the  slaughter  house  and 
normal  NaOH  solution.  Mix  30  parts  of  this  alkaline  blood  mixture  with  70  parts 
of  hot  3%  nutrient  agar.  The  poured  plates  should  be  left  half  open  over  night  in 
the  incubator  otherwise  even  cholera  will  not  grow  on  the  plates. 

Krumwiede  has  as  a  formula  for  his  medium  equal  parts  of  whole  egg  and  water, 
to  which  50%  water  egg  mixture  is  added  an  equal  amount  of  12^%  crystal  sodium 
carbonate  solution.  This  alkaline  egg  mixture  is  steamed  for  twenty  minutes.  To 
prepare  add  30  parts  of  this  alkaline  egg  mixture  to  70  parts  of  meat  extract  free 
3%  agar.  (No  meat  extract;  only  peptone  and  salt.)  The  cholera  colony  has  a 
hazy  look,  like  a  little  wad  of  absorbent  cotton  sticking  to  the  surface  with  a  metallic 
luster  halo. 

Other  selective  media  for  cholera  are  those  of  Kabeshima  in  which  a  haemoglobin 
extract  is  treated  with  alkalis  and  added  to  agar. 

The  medium  of  Esch  has  been  highly  recommended.  It  is  easy  to  make.  Heat 
500  grams  chopped  up  beef  with  250  c.c.  normal  NaOH  solution  in  a  pot  and  when 
disintegrated  filter  through  cloth  and  sterilize.  About  i  part  of  this  alkaline  extract 
is  added  to  2^  to  2  parts  of  agar.  The  plates  must  be  dry.  The  transparency  of 
this  medium  is  an  advantage. 


N.   N.   N.  MEDIUM  35 

RUSSELL'S  DOUBLE  SUGAR  AGAR 

A  fairly  stiff  agar  (2  to  3%)  with  a  reaction  of  about  plus  0.7  is  colored  with  litmus 
solution  to  produce  a  distinct  purple- violet  color.  It  may  be  necessary  to  add  more 
alkali.  To  this  litmus  tinted  agar  is  added  i%  of  lactose  and  0.1%  of  glucose  and 
the  medium  as  thus  prepared  is  tubed  and  slanted.  Sterilization  should  be  carried 
on  in  the  Arnold,  on  two  successive  days,  as  the  autoclave  temperatures  tend  to 
break  up  the  sugars. 

On  these  slants  typhoid  shows  a  delicate  growth  on  the  violet  slant  with  a  deep 
pink  in  the  butt  of  the  tube.  The  paratyphoids  show  gas  bubbles  in  a  pink  butt  with 
a  violet  slant. 

The  colon  bacillus  turns  both  slant  and  butt  a  deep  pink  and  the  butt  is  filled 
with  gas  bubbles.  To  inoculate  this  medium  we  take  material  from  a  suspicious 
colony  grown  on  Endo  and  smear  the  material  on  the  slant;  then  with  the  same  plati- 
num needle  we  stab  into  the  butt. 

Culture  Media  for  Protozoa 

MEDIUM  OF  MUSGRAVE  AND  CLEGG 

Dissolve  in  1000  c.c.  of  water  0.3  to  0.5  gram  Liebig's  extract  and  0.3  to  0.5 
gram  of  common  salt.     If  desired  for  plating  add  2  to  3%  of  agar. 
A  very  satisfactory  substitute  is  ordinary  nutrient  bouillon  diluted  one  to  ten. 

MEDIUM  OF  SMITH 

Glucose  i.o  gram;  Peptone  i.o  gram;  Nad  0.2;  Aqua  destill.  1000.0;  Na2CO3  0.3. 
Agar  q.  s.  is  added  for  solid  medium. 

MEDIUM  OF  CASTELLANI 

This  is  an  aqueous  medium  containing  i%  of  lactose  and  10%  of  egg  albumin. 
This  may  replace  water  of  condensation  in  an  agar  slant. 

AUTOLYZED   TISSUE   MEDIA 

Couret  and  Walker  have  possibly  grown  amoebae  from  liver  abscess  on  autolyzed 
tissue  media.  Sterile  organs  from  healthy  animals  as  well  as  human  placenta  are 
placed  in  sterile  flasks  and  kept  in  a  thermostat  at  4o°C.  for  about  two  weeks.  The 
liquid  from  the  autolyzed  tissues  should  be  about  neutral  and  is  either  applied  to 
the  surface  of  agar  slants  or  is  mixed  with  melted  agar.  The  medium  is  then 
sterilized. 

Now  MACNEAL  MEDIUM 

Cover  125  grams  of  chopped  up  beef  with  1000  c.c.  of  water  and  place  over  night 
in  the  refrigerator.  Strain  and  add  20  grams  of  peptone,  5  grams  salt,  10  c.c.  of 


36  CULTURE   MEDIA 

normal  sodium  carbonate  solution  and  20  to  25  grams  agar.  Prepare  as  for  nutrient 
agar  and  sterilize.  To  i  part  of  this  one-quarter  strength  meat  infusion  nutrient 
agar,  when  melted  and  cooled  down  to  6o°C.,  add  twice  its  volume  of  defibrinated 
rabbit's  blood.  This  medium  is  the  standard  one  for  the  culture  of  certain  trypano- 
somes  and  other  protozoa.  Under  the  designation  N.N.N.  medium  (Nicolle 
Novy  MacNeal)  Nicolle  has  modified  the  medium  so  that  there  is  only  salt  and  agar 
in  the  base  to  which  the  blood  is  added  instead  of  one  containing  meat  extract  and 
peptone.  It  is  the  Hb  which  seems  essential  in  the  culture  of  various  protozoa. 
Rogers  used  citrated  salt  solution,  which  was  slightly  acidified  with  citric  acid,  in 
his  culturing  of  Leishmania  from  the  splenic  blood  of  cases  of  kala  azar.  Incubation 
at  22°C. 

ROW'S   H^EMOGLOBINIZED   SALINE   MEDIUM 

Take  10  c.c.  blood  from  rabbit's  heart  or  arm  vein  of  man,  defibrinate  the  blood 
and  then  add  10  volumes  of  distilled  water  to  lake  the  cells  (liberation  of  Hb).  One 
volume  of  this  laked  blood  solution  is  added  to  two  volumes  of  sterile  1.2%  salt 
solution. 

CULTURE  MEDIA  FOR  TREPONEMATA 

I.  Noguchi  formerly  first  inoculated  material  containing  treponemata  into  the 
testicle  of  rabbits,  obtaining  by  this  procedure  a  pure  culture,  after  a  few  transfers 
to  the  testicles  of  other  rabbits.  He  now  grows  the  organism  directly  from  serum 
from  a  chancre.  Test-tubes  2  by  20  cm.  are  filled  with  15  c.c.  of  a  medium  consisting 
of  2  parts  of  2%  slightly  alkaline  agar  to  which  when  melted  and  cooled  down  to 
So°C.  is  added  i  part  of  ascitic  or  hydrocele  fluid.  At  the  bottom  of  the  medium 
in  the  tube  is  placed  a  fragment  of  fresh  sterile  tissue,  preferably  a  piece  of  rabbit's 
kidney  or  testicle.  After  the  medium  solidifies  a  layer  of  sterile  paraffin  oil  is  run 
in  so  that  it  covers  the  solid  medium  to  a  depth  of  3  cm.  The  material  is  inoculated 
at  the  bottom  of  the  tube  with  a  capillary  pipette.  Incubation  at  37°C.  is  carried 
on  for  two  weeks.  The  tissue  acts  by  removing  any  oxygen  that  may  be  present  in 
the  depths  of  the  medium.  Anaerobiosis  is  a  necessary  condition.  Many  specimens 
of  ascitic  fluid  are  unsuited.  The  tubes  of  Noguchi  and  Bronfenbrenner  are  shown 
in  Fig.  6.  Bronfenbrenner  uses  a  iH%  a8ar  instead  of  the  2%  used  by  Noguchi. 

M'Leod  and  Soga  have  simplified  Noguchi's  method  as  follows:  Take  a  test-tube 
and  fit  a  perforated  rubber  stopper  which  can  be  pushed  down  the  tube.  A  piece 
of  glass  tubing  is  passed  through  the  stopper  to  project  slightly  into  the  test-tube. 
The  other  end  of  the  glass  tube  is  drawn  out  into  a  capillary  tube  and  bent  over  at 
an  acute  angle.  The  test-tube  is  filled  to  K  or  %  of  its  depth  with  neutral  bouillon. 
This  is  freshly  boiled  and  when  cool  a  piece  of  sterile  tissue  is  dropped  in.  A  strip 
of  sterile  gauze  is  drawn  through  a  glass  bead  and  soaked  in  the  material  it  is  desired 
to  culture  and  dropped  into  the  bottom  of  the  tube  alongside  the  fragment  of  sterile 
tissue.  Ascitic  fluid  is  then  run  in  to  a  point  which  would  be  reached  by  the  bottom 
of  the  rubber  stopper.  As  quickly  as  possible  push  in  the  stopper  and  when  the 
fluid  appears  in  the  capillary  tube  seal  off  the  end  in  a  small  flame.  Material  for 
study  can  be  obtained  afterward  by  breaking  off  the  capillary  tip  and  introducing 
a  capillary  pipette. 


CULTURING  TREPONEMATA  37 

II.  Serum  Agar  of  Muhlens  and  Hofmann. — Fill  sterile  test-tubes  one-third  full 
with  horse  serum.  This  is  sterilized  on  three  successive  days  at  S5°C.  Then 
add  an  equal  amount  of  a  3%  agar  containing  0.5%  glucose  which  has  been  melted 
down  and  cooled  to  5o°C.  The  mixed  serum  agar  is  then  kept  at  S5°C.  for  two  hours. 
Such  tubes  are  inoculated  as  for  ascitic  agar  rabbit  tissue  media  and  incubated  under 
anaerobic  conditions,  preferably  in  a  flask  from  which  the  air  has  been  exhausted  and 
the  remaining  oxygen  absorbed  as  shown  in  the  anaerobic  bottle  described  and 
illustrated  in  Fig.  8. 


CHAPTER  III 
STAINING  METHODS 

IN  order  to  study  a  bacterial  or  blood  specimen  the  first  essential  is 
a  properly  prepared  film;  the  matter  of  staining  is  of  less  importance. 

The  slide  or  cover-glass,  after  cleaning  with  soap  and  water  or  by  special  solutions, 
should  be  polished  with  a  piece  of  old  linen.  If  a  glass  surface  is  free  of  grease  a 
loopful  of  water  will  smear  out  evenly  and  over  the  entire  surface.  The  only  quick 
practical  way  to  make  the  slide  or  cover-glass  grease  free  is  to  burn  the  surface  for  a 
moment  in  a  Bunsen  or  alcohol  flame.  The  cover-glass  must  not  be  warped.  To 
make  a  preparation,  apply  a  small  loopful  of  distilled  water  on  the  slide  or  cover-glass 
and,  touching  a  colony  with  a  platinum  needle,  stir  the  transferred  culture  into  the 
loopful  (not  drop)  of  water.  The  mistake  is  almost  invariably  made  of  taking  up 
too  much  bacterial  growth.  Fluid  cultures  do  not  need  dilution.  Smearing  the 
mixture  over  a  large  part  of  the  cover-glass  or  over  an  equal  area  of  a  slide,  it  is 
allowed  to  dry.  If  very  little  water  is  used,  the  preparation  dries  readily.  Other- 
wise it  can  be  dried  in  the  fingers  high  over  a  flame.  As  soon  as  dry,  the  cover-glass 
should  be  passed  three  times  through  the  flame,  film  side  up,  to  fix  the  preparation. 
Slides  may  be  fixed  by  passing  them  five  times  through  the  flame,  but  the  method  by 
burning  alcohol  recommended  for  fixing  blood-films  gives  more  satisfactory  bacterial 
fixation.  For  routine  work  the  stain  recommended  is  a  dilute  carbol  fuchsin.  Drop 
about  5  to  10  drops  of  water  on  the  cover-glass,  then  add  i  drop  of  carbol  fuchsin. 
Allow  the  dilute  stain  to  act  from  one  to  two  minutes,  then  wash  in  water,  dry  be- 
tween small  squares  of  filter-paper  (4X4  inches),  and  mount  in  balsam  or  the  oil 
used  for  the  K 2-inch  immersion  objective.  LofBer's  methylene  blue  is  equally  good 
as  a  stain. 

By  far  the  best  mounting  medium  is  liquid  petrolatum.  This  not  only  has  the 
advantage  of  always  being  of  proper  consistence  for  mounts,  as  opposed  to  Canada 
balsam,  which  must  frequently  be  made  thinner  with  xylol,  but  it  is  less  sticky  and 
does  not  develop  the  acidity  which  causes  balsam  mounts  of  Romanowsky  stains 
to  fade.  Furthermore,  it  has  superior  optical  qualities.  It  is  also  applicable  for 
mounting  small  insects  and  sporangia  of  moulds.  For  permanent  preparations  the 
border  of  the  cover-glass  should  be  sealed  with  gold  size  or  some  other  cement. 
Some  prefer  to  mount  directly  in  water  without  preliminary  drying.  It  is  good 
practice  to  make  a  rule  to  always  keep  the  smeared  side  of  the  preparations  up — 
never  allowing  it  to  be  reversed.  By  this  simple  rule,  preparations  can  be  carried 
through  the  most  complicated  staining  methods  without  the  necessity  of  scratching 
the  cover-glass,  etc.,  to  see  which  is  the  film  side.  In  grasping  a  cover-glass  with  a 
Cornet  or  Stewart  forceps,  be  sure  that  the  tips  are  well  by  the  margin  of  the  glass, 
otherwise  the  stain  will  drain  off.  In  staining  with  slides,  the  grease  pencil  and  the 

38 


GRAM'S  STAINING  METHOD  39 

glass  tubing,  as  recommended  under  Blood  Smears,  will  be  found  useful.  The  dilute 
carbol  fuchsin  and  Loffler's  methylene  blue  are  probably  the  best  routine  stains.  As 
a  rule  better  preparations  are  obtained  with  dilute  stains  than  with  more  concen- 
trated ones. 

Loffler's  Alkaline  Methylene  Blue.— Saturated  alcoholic  solution 
of  methylene  blue,  30  c.c.;  i  to  10,000  caustic  potash  solution,  100 
c.c.  (Two  drops  of  a  10%  solution  KOH  in  100  c.c.  of  water  makes  a 
i  :  10,000  solution.) 

Carbol  Fuchsin  (Ziehl-Neelsen).— Saturated  alcoholic  solution  basic 
fuchsin,  10  c.c.;  5%  aqueous  solution  carbolic  acid,  100  c.c. 

Gram's  Method. — The  most  important  staining  method  in  bacteri- 
ological technic  and  the  one  so  rarely  giving  satisfactory  results  to  the 
inexperienced  is  Gram's  stain.  In  using  this  method,  the  following 
points  must  be  kept  in  mind: 

1.  Laboratory  cultures   (subcultures)   which  have  been  carried  over  for  years 
frequently  lose  their  Gram  characteristics. 

2.  Cultures  which  are  several  days  old  or  dead  or  degenerated  do  not  stain  char- 
acteristically. 

3.  The  aniline  gentian  violet  deteriorates  when  exposed  to  light  in  two  or  three 
days — it  should  be  kept  in  the  dark.     It  should  have  a  rich,  creamy,  violet  appear- 
ance. 

4.  The  iodine  solution  deteriorates  and  becomes  light  in  color.     It  should  be  of 
a  rich  port-wine  color. 

5.  The  decolorizing  with  95%  alcohol  should  stop  as  soon  as  no  more  violet  stain 
streams  out.     This  is  best  observed  over  a  white  background,  washing  at  intervals. 
Do  not  confuse  stain  on  forceps  for  that  on  preparation. 

6.  The  preparation  should  be  thin  and  evenly  spread.     Some  prefer  carbol 
gentian  violet  to  aniline  gentian  violet.     (Saturated  alcoholic  solution  of  gentian 
violet,  i  part;  5%  aqueous  solution  of  carbolic  acid,  10  parts.)     This  tends  to  over- 
stain. 

The  formula  for  aniline  gentian  violet  is  i  part  of  saturated  alcoholic  solution 
gentian  violet  and  3  parts  of  aniline  oil  water  (made  by  adding  2  c.c.  aniline  oil  to 
100  c.c.  distilled  water,  shaking  violently  for  three  to  five  minutes  and  then  filtering 
several  times  to  get  rid  of  the  objectionable  oil  droplets  which,  in  a  Gram-stained 
preparation,  show  as  confusing  black  dots). 

The  following  stock  solutions  of  Weigert  are  recommended: 

No.  i  No.  2. 

Gentian  violet. ......     2  grams.  Gentian  violet.  ...       2  grams. 

Aniline  oil 9  c.c.  Distilled  water —   100  c.c. 

Alcohol  (95%) 33  c.c. 

These  stock  solutions  keep  indefinitely.  Mix  i  c.c.  of  No.  i  with  9  c.c.  of  No.  2. 
Filter.  This  keeps  about  two  weeks  and  is  the  solution  to  pour  on  the  preparation. 
It  may  be  kept  on  from  two  to  five  minutes.  Some  hasten  the  staining  by  steaming 


STAINING   METHODS 


as  for  tubercle  bacilli.  Next  wash  the  preparation  with  water  and  flood  the  cover- 
glass  with  Gram's  iodine  solution.  Some  bacteriologists  simply  pour  off  excess  of 
aniline  gentian  violet  and  immediately  drop  on  the  iodine  solution.  It  is  well  to 
repeat  the  application  of  the  iodine  solution  a  second  time..  The  iodine  solution  is 
left  on  one  minute  or  until  the  preparation  has  a  coffee-grounds  color. 

GRAM'S  IODINE  SOLUTION 

Iodine i  gram. 

Potassium  iodide 2  grams. 

Distilled  water 300  c.c. 

After  washing  off  the  excess  of  iodine  solution  at  the  tap,  drop  on  95%  alcohol 
and  decolorize  until  no  more  violet  color  streams  out.  Now  wash  again  and  counter- 
stain  either  with  the  dilute  carbol  fuchsin  or  with  a  saturated  aqueous  solution  of 
Bismark  brown. 

The  Gram-positive  bacteria  are  stained  a  deep  violet. 

In  staining  smears  of  pus  for  gonococci  or  other  Gram-negative  bacteria  it  is  best 
to  first  stain  with  the  gentian-violet  solution  for  two  to  five  minutes.  Then  wash 
and  examine  the  preparation  mounted  in  water.  The  organisms  stand  out  promi- 
nently. After  noting  the  presence  of  the  cocci  treat  the  smear  with  the  Gram 
solution  and  proceed  as  in  the  usual  Gram  staining  technic. 


STAINED  BY  GRAM'S  METHOD 
S.  pyogenes  aureus. 
S.  pyogenes  albus. 
S.  pyogenes. 
M.  tetragenus. 
Pneumococcus. 
Anthrax  bacillus. 
Tubercle  bacillus. 
Lepra  bacillus. 
Tetanus  bacillus. 
Diphtheria  bacillus. 
B.  aerogenes  capsulatus. 
Oidium  albicans. 
Mycelium  of  actinomyces. 
Saccharomyces. 
Hofmann's  bacillus. 
B.  xerosis. 


NOT  STAINED  BY  GRAM'S  METHOD 
Meningococcus. 
M.  catarrhalis. 
M.  melitensis. 
B.  typhosus. 
B.  coli  communis. 
B.  dysenteriae  (Shiga). 
Sp.  cholerae  asiaticae. 
B.  pyocyaneus. 
B.  mallei. 

B.  pneumonias  (Friedlander). 
B.  proteus. 
B.  of  influenza. 
B.  of  bubonic  plague. 
B.  of  chancroid. 
B.  of  Koch- Weeks. 
Gonococcus. 


Practically  all  pathogenic  cocci  are  Gram-positive,  except  the  Gonococcus,  the 
Meningococcus ',  the  M .  catarrhalis,  and  the  M.  melitensis. 

Practically  all  pathogenic  bacilli  are  Gram-negative,  except  the  spore-bearing 
ones  (exception  B.  malig.  cedemat.},  the  acid-fast  ones,  diphtheria  and  diphtheroid 
organisms. 

The  bacillus  of  glanders  is  Gram-negative. 


ACID-FAST  STAINING  41 

Method  for  Staining  Acid-fast  Bacilli. — i.  Carbol  fuchsin,  with  gen- 
tle steaming  for  three  to  five  minutes  or  in  the  cold  for  fifteen  minutes. 

2.  Wash  in  water. 

3.  Decolorize  in  95%  alcohol  containing  3%  of  hydrochloric  acid 
(acid  alcohol),  until  only  a  suggestion  of  pink  remains — almost  white. 

4.  Wash  in  water. 

5.  Counters  tain  in  saturated  aqueous  solution  of  methylene  blue  or 
with  Loffler's  methylene  blue. 

6.  Wash,  dry,  and  mount. 

The  steaming  of  the  slides  with  carbol  fuchsin  is  most  conveniently  carried  out 
by  resting  the  slides  on  a  piece  of  glass  tubing  bent  into  a  V  or  U  shape. 

The  Leprosy  Bacillus  is  usually  considered  as  being  rather  easily  decolorized  by 
alcohol.  It  is  therefore  often  recommended  to  use  20%  aqueous  solution  of  sul- 
phuric acid  or  nitric  acid  for  decolorization  instead  of  the  acid  alcohol  above  recom- 
mended for  tubercle  bacilli.  I  have  often  found  the  leprosy  bacilli  as  resistant  to 
alcohol  as  tubercle  bacilli.  The  smegma  bacillus,  however,  easily  decolorizes  with 
the  acid  alcohol  and  in  a  well-decolorized  smear  from  urinary  sediment  one  can 
usually  feel  sure  that  any  acid-fast  bacilli  are  tubercle  bacilli. 

Fontes  Method. — A  method  in  which  the  organisms  or  granules  which  stain  by 
the  Gram  method,  and  to  which  so  much  importance  is  attributed  by  Much,  may 
be  stained,  as  well  as  those  retaining  acid-fast  properties,  has  been  proposed  by 
Fontes.  The  method  is  to  stain  the  preparation  with  carbol  fuchsin,  decolorize 
with  acid  alcohol,  then  carry  through  the  various  steps  of  the  Gram  method, 
counterstaining,  however,  with  Bismark  brown.  Fontes  in  his  method  used  i  part 
of  absolute  alcohol  and  2  parts  of  acetic  acid  as  the  decolorizing  agent.  I  have 
obtained,  however,  just  as  satisfactory  results  with  the  acid  alcohol.  By  this 
method  the  acid-fast  tubercle  bacilli  show  as  red  rods  dotted  with  violet  granules. 
Those  which  do  not  fully  retain  acid-fast  properties  show  as  zigzag  violet  lines. 

Herman's  Stain  for  Tubercle  Bacilli. — It  has  been  claimed  that  this  stain  gives 
better  satisfaction  than  the  Ziehl-Neelsen.  It  consists  of  two  solutions:  (i)  am- 
monium carbonate  in  distilled  water,  i%;  (2)  crystal  violet  (methyl  violet  6B)  in 
95%  ethyl  alcohol,  3%.  The  two  solutions  are  kept  in  separate  bottles  and,  for 
staining,  i  part  of  (2)  is  mixed  with  3  parts  of  (i).  The  sections  are  placed  on  a 
cover-glass,  the  water  evaporated,  and  about  7  drops  of  the  staining  mixture 
are  placed  on  the  specimen  and  allowed  to  steam  for  one  minute  over  a  water-bath. 
Place  for  a  few  seconds  in  10%  nitric  acid  and  then  in  95%  alcohol  to  decolorize. 
Mount  without  a  counterstain  or  use  eosin  i%  or  a  very  dilute  fuchsin.  The  organ- 
isms are  purple.  This  staining  method  may  be  applied  to  smears  of  concentrated 
or  unconcentrated  sputum  in  the  same  manner  as  for  sections  of  tissue. 

Smith's  formol  fuchsin : 

Saturated  alcoholic  solution  basic  fuchsin 10  c.c. 

Methyl  alcohol 10  c.c. 

Formalin 10  c.c. 

Distilled  water  to  make 100  c.c. 


STAINING   METHODS 


This  gives  a  very  sharp  differentiation  of  bacteria  and  nuclear  structures.  It 
has  a  purplish  tinge.  Fixation  by  heat  gives  the  best  staining.  Allow  the  stain  to 
act  for  two  to  ten  minutes.  It  should  not  be  used  until  after  standing  twenty-four 
hours,  and  after  standing  about  two  weeks  it  appears  to  lose  its  sharp  staining  power 

Archibald's  Stain. — This  is  an  excellent  bacterial  stain  and  has  been 
highly  recommended  by  Blue  and  McCoy  in  plague  work. 


SOLUTION  No.  i. 

Thionin 0.5 

Phenol  crys 2.5 

Formalin i.o 

Water 100.0 


SOLUTION  No.  2. 

Methylene  blue 0.5 

Phenol  crys 2.5 

Formalin i.o 

Water 100.0 


Dissolve  for  twenty-four  hours.     Mix  equal  parts  and  filter.     Stain  smears  fixed 
by  heat  or  otherwise  for  ten  seconds. 

Nicolle's  Carbol  Thionin 

Sat.  sol.  thionin  in  50%  alcohol 


Carbolic  acid  solution  (2%). 


10  c.c. 
100  c.c. 


Pappenheim's  Stain. — Take  a  very  small  portion  of  methylene  green  on  the  point 
of  a  penknife  and  shake  it  into  a  test-tube.  Then  take  up  twice  as  much  pyronin  and 
deposit  it  in  the  same  test-tube.  Fill  the  test-tube  one-half  full  with  water  and  the 
solution  should  have  a  distinct  reddish-violet  color.  A  drop  on  a  piece  of  filter-paper 
shows  a  violet  center  and  peripheral  green  ring.  The  solution  should  be  fresh. 
Stain  from  two  to  five  minutes.  Differentiate  with  a  little  resorcin  on  a  penknife 
point  dissolved  in  one-quarter  of  a  test-tube  full  of  alcohol.  Dehydrate,  clear  and 
mount.  Polymorphonuclear  nuclei  stain  greenish;  nuclei  of  mononuclears  and 
plasma  cells  from  bluish-red  to  dull  violet.  Cytoplasm  of  lymphocytes  and  plasma 
cells  purplish-red.  Bacteria  red. 

Romanowsky  Stains. — See  under  section  on  Blood.  For  mounting 
specimens  showing  chromatin  staining,  as  malarial  parasites,  trypano- 
somes,  intestinal  flagellates,  etc.,  liquid  petrolatum  is  to  be  highly  rec- 
ommended. The  chromatin  staining  lasts  without  any  fading  for  at 
least  two  years.  The  acidity  of  balsam  causes  rapid  fading  of  the 
chromatin. 

Neisser's  Stain  for  Diphtheria  Bacilli 
SOLUTION  No.  i                           SOLUTION  No.  2 
Methylene  blue. ...  o.  i  gram.     Bismark  brown 


0.2 


Alcohol 2  c.c. 

Glacial  acetic  acid. .       5  c.c. 

Distilled  water 95  c.c. 

Dissolve  the  methylene  blue 
in  the  alcohol  and  add  it  to 
the  acetic  acid  water  mixture. 
Filter. 


Water  (boiling) 100  c.c. 

Dissolve  the  stain  in  the  boil- 
ing water  and  filter. 


DIPHTHERIA  STAINING  43 

To  stain:  Fix  the  preparation.  Pour  on  the  dilute  acetic  acid  methylene  blue 
solution  and  allow  to  act  from  thirty  to  sixty  seconds.  Wash.  Then  pour  on  the 
Bismark-brown  solution,  and  after  thirty  seconds  wash  off  with  water.  Dry  and 
mount.  The  bodies  of  the  bacilli  are  brown  with  dark  blue  dots  at  either  end. 

Neisser  recommends  only  five  seconds  as  the  time  of  application  of  each  solution. 
He  also  recommends  that  the  culture  be  only  nine  to  eighteen  hours  old  and  that  the 
temperature  of  the  incubator  shall  not  exceed  36°C.  Incubation  at  37°C.  gives 
satisfactory  results. 

Ponder's  Stain  for  Diphtheria  Bacilli 

Toluidin  blue  (Grubler) o. 02  gram. 

Glacial  acetic  acid i  c.c. 

Absolute  alcohol 2  c.c. 

Distilled  water  to 100  c.c. 

The  film  is  made  on  a  cover-glass  and  fixed  in  the  usual  way.  A  small  quantity 
of  the  stain  is  spread  on  the  film  and  the  cover-glass  is  turned  over  and  mounted  as 
a  hanging-drop  preparation.  The  metachromatic  granules  of  the  diphtheria  bacilli 
stain  with  striking  intensity.  With  diphtheroids,  the  more  intense  staining  sharply 
differentiates  from  ordinary  cocci  and  bacilli,  which  show  in  the  preparation  only  as 
faint  light  blue  bodies.  It  is  a  most  excellent  stain  for  bringing  out  the  ascopores  of 
yeasts.  In  my  opinion  the  stain  is  more  valuable  than  the  Neisser  method. 

Capsule  Staining. — The  best  method  for  studying  bacteria,  as  to 
presence  of  capsules,  is  in  the  hanging  drop,  with  the  greater  part  of  the 
light  shut  off  by  the  diaphragm. 

In  material  where  capsules  are  well  developed,  as  in  pneumonic  sputum,  the 
Gram  method  of  staining  brings  out  the  capsule  perfectly.  This  is  of  diagnostic 
value,  as  the  more  or  less  nonpathogenic  pneumococci  common  about  the  mouth 
do  not  seem  to  show  a  capsule  when  stained  in  this  way.  The  India  ink  method  of 
staining  gives  good  results  for  capsules. 

The  most  beautiful  method  of  staining  capsules  is  the  latest  one  pro- 
posed by  Muir. 

1.  Prepare  thin  film,  dry  and  stain  in  carbol  fuchsin  one-half  minute;  the  prepa- 
ration being  gently  heated  (steamed). 

2.  Wash  slightly  in  95%  alcohol,  then  wash  well  afterward  in  water. 

3.  Flood  preparation  in  mordant  for  five  to  ten  seconds. 

Mordant. — Sat.  aqueous  sol.  mercuric  chloride 2  parts. 

Tannic  acid  (20%  aqueous  sol.) 2  parts. 

Sat.  aqueous  sol.  potash  alum 5  parts. 

4.  Wash  in  water  thoroughly. 

5.  Treat  with  95%  alcohol  for  one  minute.     (The  preparation  should  have  a  pale 
red  color.) 

6.  Wash  well  in  water. 

7.  Counterstain  with  methylene  blue  one-half  minute. 


44  STAINING   METHODS 

8.  Dehydrate  in  alcohol.  Clear  in  xylol  and  mount.  (May  simply  dry  specimen 
with  filter-paper.) 

Rosenow'a  Capsule  Stain. — Make  a  very  thin  smear  of  the  pathological  material 
and  when  nearly  dry  cover  the  preparation  for  ten  to  twenty  seconds  with  10% 
tannic  acid  solution.  Wash  in  water  and  blot.  Stain  with  aniline  gentian  violet 
by  gently  steaming  for  one-half  to  one  minute.  Wash  in  water.  Apply  Gram's 
iodine  solution  for  one-half  to  one  minute.  Decolorize  in  95%  alcohol  and  then 
stain  with  alcoholic  solution  of  eosin.  Wash  in  water,  dry  and  mount. 

Flagella  Staining. — Inoculate  a  tube  of  sterile  water  (gently)  in 
upper  part,  with  just  enough  of  an  eighteen  to  twenty-four-hour-old 
agar  culture  to  produce  faint  turbidity.  Incubate  for  two  hours  at 
37°C.  From  the  upper  part  of  culture  take  a  loopful  and  deposit  it  on 
a  cover-glass.  Dry  in  thermostat  for  one  to  five  hours  or  over  night. 
Use  perfectly  clean  cover-glasses.  To  stain  by 

Muir's  Modified  Pitfield  Method 

1.  Flood  specimen  with  mordant.     Steam  gently  one  minute. 

Mordant. — Tannic  acid  (10%  aqueous  solution) 10  c.c. 

Sat.  aq.  sol.  mercuric  chloride 5  c.c. 

Sat.  aq.  sol.  alum 5  c.c. 

Carbol  fuchsin 5  c.c. 

Allow  precipitate  to  settle  or  centrifuge.     Keeps  only  one  week. 

2.  Wash  well  in  water  for  two  minutes. 

3.  Dry  carefully — preferably  in  incubator. 

4.  Pour  on  stain.     Steam  gently  one  minute. 

Stain. — Sat.  aq.  sol.  alum 10  c.c. 

Sat.  ale.  sol.  gentian  violet 2  c.c. 

(May  use  carbol  fuchsin  instead  of  gentian  violet.) 
Stain  only  keeps  two  days. 

5.  Wash  well  in  water.     Dry  and  mount. 

Zettnow's  Flagella  Staining  Method 

Solution  I. — Dissolve  2  grams  of  tartar  emetic  in  40  c.c.  water. 

Solution  II. — Dissolve  10  grams  tannin  in  200  c.c.  water.  To  the  200  c.c.  solution 
II,  warmed  to  50  or  6o°C.,  add  30  c.c.  of  the  tartar  emetic  solution.  The  turbidity 
of  the  mordant  should  entirely  clear  up  on  heating.  The  mordant  should  keep  for 
months  when  a  small  crystal  of  thymol  is  added  to  it. 

Next  dissolve  i  gram  silver  sulphate  in  250  c.c.  distilled  water.  Of  this  solution 
take  50  c.c.  and  add  to  it  drop  by  drop  ethylamine  (this  comes  in  a  33%  solution) 
until  the  yellowish-brown  precipitate  which  forms  at  first  is  entirely  dissolved  and 
the  fluid  is  entirely  clear.  It  requires  only  a  few  drops.  The  bacterial  preparations 
prepared  as  described  above  are  floated  in  a  little  mordant  contained  in  a  Petri  dish 


STAINING  PROTOZOA  45 

which  is  heated  over  a  water-bath  for  five  to  seven  minutes.  Take  the  dish  contain- 
ing the  preparation  off  the  water-bath  and  as  soon  as  it  becomes  slightly  opalescent 
as  the  result  of  cooling  remove  the  cover-glass  preparation  and  wash  thoroughly  in 
water.  Then  heat  a  few  drops  of  the  ethylamine  silver  solution  upon  the  mordanted 
cover  preparation  until  it  just  steams  and  the  margin  appears  black.  Next  wash 
thoroughly  in  water  and  mount.  This  gives  the  most  satisfactory  results  of  any 
method  I  have  ever  experimented  with. 

Spore  Staining. —The  most  satisfactory  spore  staining  method  is 
really  the  negative  staining  of  the  spore  obtained  when  a  bacterial 
preparation  is  stained  by  dilute  carbol  fuchsin  or  Loffler's  methylene 
blue.  The  spore  appears  as  a  highly  refractile  piece  of  glass  in  a  colored 
frame. 

The  acid-fast  method,  as  for  tubercle  bacilli,  gives  good  results.  The  decoloriz- 
ing, however,  must  be  lightly  done,  otherwise  the  spore  will  lose  its  red  stain. 

M  oiler's  Method. — Fix  films  and  then  treat  with  chloroform  for  one  or  two 
minutes.  Wash  thoroughly  and  treat  with  a  5%  solution  chromic  acid  for  one 
minute.  Wash  in  water  and  then  stain  as  for  acid-fast  organisms  with  carbol 
fuchsin.  Use  a  i%  sulphuric  acid  solution  instead  of  the  3%  acid  alcohol. 

Agar  Jelly  Staining  Method  of  H.  C.  Ross 

Very  clear  i^%  solution  of  agar  is  colored  with  Unna's  polychrome  methylene 
blue,  Giemsa's  solution,  thionin  or  Gram's  solution  of  iodine.  Very  thin  smears  of 
blood,  faeces  or  gastric  content  sediment  are  made  and  either  fixed  lightly  in  the  flame 
or  air  dried.  A  drop  of  the  melted  colored  agar  solution  is  placed  on  the  smeared 
cover-glass  and  this  is  mounted  immediately  on  a  clean  slide.  The  preparation  is 
ready  for  examination  in  about  two  minutes. 

The  Staining  of  Protozoa 

Unless  staining  albuminous  material  it  is  well  to  add  a  little  blood- 
serum  albumin  fixative  or  white  of  egg  to  the  preparation — about  one 
loopful  to  a  smear.  The  serum  or  white  of  egg  is  best  preserved  by 
the  addition  of  2  %  chloroform  and  kept  tightly  corked. 

Giemsa's  Method. — Fix  moist  smears  with  a  fixative  made  by  adding  i  part  of  95% 
alcohol  to  2  parts  of  saturated  aqueous  solution  of  bichloride  of  mercury.  Keep  in 
this  solution  i  to  12  hours.  Now  wash  for  a  few  seconds  in  water  and  then  for  about 
five  minutes  with  a  dilute  Lugol's  solution  (KI,  2  gm.;  Lugol's  solution,  3  c.c.;  Aqua, 
100  c.c.).  Now  wash  in  water  and  then  in  a  0.5%  solution  of  sodium  thiosulphate 
to  remove  the  iodine  which  was  used  to  remove  the  mercury.  Wash  in  water  five 
minutes,  then  stain  with  Giemsa's  stain  as  used  in  blood-work  for  one  to  ten  hours. 
Wash  and  mount. 

Vital  Staining  of  Protozoa  with  Neutral  Red  Solution. — As  a  stock 
solution  one  uses  a  0.5%  aqueous  solution  of  neutral  red. 


46  STAINING  METHODS 

The  drop  of  salt  solution  or  water  on  the  slide  should  be  tinged  a  light  violet-rose 
color  with  a  fraction  of  a  loopful  and  the  faeces  or  other  material  emulsified  in  this. 

Protozoa  take  a  rose-pink  color  with  a  distinct  differentiation  be- 
tween endoplasm  and  ectoplasm. 

Should  the  faeces  be  quite  alkaline  the  neutral  red  will  be  decomposed  with  the 
formation  of  bilirubin-like  crystals. 

The  Giemsa  formalin  method  described  under  Blood-work  is  of  value  in  certain 
cases. 

Panoptic  Method. — Highly  to  be  recommended  for  the  staining  of  protozoa, 
whether  in  smears  or  in  sections,  is  the  Panoptic  method. 

1.  Wright's  or  Leishman's  stain  for  one  minute. 

2.  Dilute  with  water  and  allow  dilute  stain  to  act  for  three  to  ten  minutes.     Wash 
in  water  and  then 

3.  Pour  on  dilute  Giemsa's  stain.     Allow  to  stain  from  thirty  minutes  to  twenty- 
four  hours.     Differentiate  with  i :  1000  acetic  acid  solution  until  blue  stain  just 
shows  commencing  diffusion  into  the  acetic  acid.     Then  wash  in  water,  95%  alcohol, 
absolute  alcohol  and  treat  with  xylol  and  mount  in  liquid  petrolatum. 

With  preparations  other  than  blood  smears,  as  sections,  it  is  better  to  go  from  95% 
alcohol  to  oil  of  origanum,  then  mount. 

Owing  to  the  great  value  of  a  sharp  nuclear  picture  in  differentiating  amoebae 
it  is  of  great  importance  to  use  some  iron  haematoxylin  method.  That  of  Weigert 
is  given  in  the  appendix. 

Mallory's  Phosphotungstic  Haematoxylin. — Fix  moist  smears,  film  surface  down, 
in  Zenker's  fluid  for  five  to  ten  minutes.  Wash  in  water,  treat  with  Gram's  solu- 
tion and  wash  with  70%  alcohol  until  all  the  yellow  color  is  discharged.  Wash  in 
water.  Then  stain  with  Mallory's  phosphotungstic  hcematoxylin  for  one-half  hour. 
Wash  clear  and  mount.  See  appendix. 

Mallory's  Differential  Stain  for  Amoebae. — Staining  in  saturated  aqueous  solution 
thionin  for  from  three  to  five  minutes.  Next  differentiate  in  2%  aqueous  solution 
oxalic  acid  for  one-half  to  one  minute.  Then  wash  in  water,  clear  and  mount. 
Nuclei  of  amoebae  are  stained  a  brownish  red. 


Rosenbusch  Iron  Haemotoxylin  Stain 

Rapidly  smear  out  with  a  toothpick  a  small  particle  of  faeces  or  other  material 
containing  protozoa  and,  while  still  moist,  fix  by  Giemsa's  method  and,  after  getting 
rid  of  the  mercury  with  iodine  followed  by  95%  alcohol,  treat  smears  with  a  3.5% 
solution  of  iron-alum  in  distilled  water  for  one-half  hour  or  over  night,  then  wash 
thoroughly  in  distilled  water. 

Then  stain  from  five  to  twenty  minutes  in  the  following  haematoxylin  stain:  (i) 
i%  solution  of  haematoxylin  in  95%  alcohol.  It  takes  at  least  ten  days  to  ripen. 
(2)  A  saturated  solution  of  lithium  carbonate.  Add  to  10  c.c.  of  the  haematoxylin 
solution  5  to  6  drops  of  the  lithium  carbonate  one.  Next  wash  well  and  differentiate 
with  about  a  i%  solution  of  the  iron  alum.  Again  wash  in  water,  pass  through 
alcohols  to  xylol  and  mount  in  balsam. 


STAINING  TREPONEMATA  47 

Mallory's  Iron  Haematoxylin  Method 

I  have  obtained  beautiful  staining  with  this  simple  method.  The  great  point  in 
technic  is  the  watching  of  the  differentiation. 

Treat  sections  or  moist  smears  fixed  by  Giemsa's  method  with  10%  aqueous  solu- 
tion of  ferric  chloride  for  three  to  five  minutes.  Then  drain  off  iron  solution,  blot 
the  section  and  stain  for  four  minutes  with  a  freshly  made  solution  of  i  %  haematoxylin 
in  water.  To  make  this  add  a  few  small  crystals  to  4  or  5  c.c.  water  in  a  test-tube 
and  dissolve  by  heat.  The  stain  deteriorates  after  twenty-four  hours.  Wash  in 
water.  Differentiate  with  a  Y±%  aqueous  solution  of  ferric  chloride.  The  dif- 
ferentiation is  complete  in  from  a  few  seconds  to  one  or  more  minutes  according  to 
depth  of  staining  and  thickness  of  film.  Wash  in  water,  pass  through  alcohols  and 
xylol  and  mount. 

Spirochaete  Staining  Method  (Fontana) 

The  smears  must  be  air  dried,  not  fixed  by  heat.  Cover  films  with  Huge's  fluid 
which  is  i  c.c.  acetic  acid,  20  c.c.  formalin  and  100  c.c.  distilled  water.  Flood  films 
several  times  with  this  fluid.  Wash  in  water  and  cover  with  a  mordant  of  5%  tannic 
acid  in  i  %  carbolic  acid  solution.  Heat  the  mordant  on  the  slide  until  steam  arises, 
and  allow  the  heated  mordant  to  act  about  thirty  seconds.  Wash  in  water  and 
without  drying  pour  on  the  silver  stain,  heat  until  steam  arises,  leave  heated  stain 
on  for  thirty  seconds,  wash  in  water,  blot,  dry  and  examine  with  immersion  objective. 
The  treponemata  are  brownish  to  black. 

To  make  the  silver  stain  make  a  J4%  solution  of  silver  nitrate  in  distilled  water. 
Then  add  drop  by  drop  ammonia  until  a  slight  turbidity  is  produced;  only  a  trace 
of  ammonia  is  required  and  any  excess  again  clears  up  the  solution  and  makes  it 
useless. 


CHAPTER  IV 

STUDY  AND  IDENTIFICATION  OF  BACTERIA— GENERAL 
CONSIDERATIONS 

IN  order  to  study  bacteria  it  is  necessary  to  isolate  them  in  pure  cul- 
ture. This  may  be  accomplished  by  taking  one  or  more  loopfuls  of 
the  material  and  mixing  it  in  a  tube  of  melted  agar  or  gelatin.  From 
this  first  tube  one  or  more  loopfuls  are  transferred  to  a  second  tube  of 
melted  agar  or  gelatin,  and  from  this  a  third  transfer  is  made,  thereby 
giving  us  tubes  in  which  the  distribution  of  the  bacteria  is  one  or  more 
hundred  times  less  in  the  second  than  in  the  first  tube,  and  equally  more 
dilute  in  the  third  than  in  the  second.  When  we  pour  the  contents 
of  the  tubes  into  Petri  dishes  we  would  have  the  bacterial  colonies  on 
the  first  plate  so  thick  that  it  would  be  impossible  to  pick  up  a  single 
colony  with  a  platinum  needle  without  touching  an  adjacent  one.  On 
the  second  plate  the  distribution  might  be  such  that  we  should  have 
discrete,  well  separated  colonies,  material  from  which  could  be  taken  up 
on  the  point  of  the  needle  or  loop  without  touching  any  other  colony. 
If  the  second  plate  did  not  meet  these  requirements,  the  third  would. 

In  clinical  bacteriology  we  work  almost  entirely  with  organisms  preferring  blood- 
heat  temperature,  hence  it  is  necessary  to  use  agar  or  blood-serum  as  standard  media 
for  the  obtaining  of  isolated  colonies.  Gelatin  is  of  little  value  for  this  purpose  in 
medical  work.  In  using  agar  it  will  be  remembered  that  it  solidifies  at  a  temperature 
slightly  below  4o°C.  and  does  not  melt  again  until  it  is  subjected  to  a  temperature 
practically  that  of  boiling.  Again,  if  the  temperature  of  the  media  exceeds  44°C.  it 
may  affect  injuriously  the  organisms  we  wish  to  study.  Consequently  it  requires 
careful  attention  and  quick  work  to  inoculate  the  tubes,  mix,  transfer  and  pour  into 
plates  within  the  limits  of  a  temperature  which  injures  the  organisms,  and  one  which 
brings  about  the  solidification  of  the  agar. 

Again,  we  not  only  have  colonies  developing  from  organisms  which 
have  been  fixed  at  the  surface  as  the  agar  solidified  in  the  plate,  but 
more  numerous  ones  developing  from  bacteria  caught  in  the  depths  of 
the  media.  Therefore  we  have  superficial  and  deep  colonies.  Except 
to  the  person  of  great  experience,  all  deep  colonies  look  alike  and  there 
is  at  times  great  difficulty  in  deciding  whether  a  colony  is  deep  or  super- 
ficial. It  is  in  the  matter  of  trying  to  obtain  information  from  the 

48 


COLONY  ISOLATION 


49 


differences  in  deep  colonies  that  the  greatest  difficulties  in  the  study 
of  bacteriology  arise.  By  using  the  method  of  simply  stroking  plates 
along  five  or  six  parallel  lines  from  one  side  of  the  plate  to  the  other 
with  a  bent  glass  rod,  platinum  loop,  or  a  small  cotton  swab,  we  obtain 
colonies  which  are  well  separated  and  which  are  entirely  superficial. 


FIG.  9. — Petri  agar  plate.  Made  by  spreading  scrapings  from  the  mouth  over 
sterilized  nutrient  agar;  after  forty-eight  hours  in  the  thermostat  the  light  "colonies" 
develop.  Streaked  plate.  (Delafield  and  Prudden.) 

We  pour  about  10  c.c.  of  agar,  blood  agar  or  Endo  media  into  Petri 
dishes  and  keep  them  in  the  refrigerator  for  immediate  use. 

Smeared  or  Stroked  Poured  Petri  Plates. — The  material  as  pus,  faeces,  throat 

membrane,  etc.,  should  be  evenly  distributed  in  a  tube  of  sterile  water  or  bouillon; 

the  swab  which  was  originally  used  for  obtaining  the  material  being  then  pressed 

against  the  sides  of  the  test-tube  to  express  excess  of  fluid  and  then  stroked  gently 

4 


50  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

over  successive  lines  on  one  plate.  Or,  if  the  organisms  be  very  abundant,  over  a 
second  plate  without  recharging  it  from  the  inoculated  tube. 

According  to  my  experience  a  very  satisfactory  method  is  to  take  a  loopful  from 
the  bouillon  tube  suspension  of  the  pus  or  faeces  and  deposit  the  fluid  in  the  platinum 
loop  on  the  left  half  of  the  poured  plate  then,  without  recharging  the  loop,  we  touch 
the  right  half  of  the  plate.  Now  taking  a  bent  glass  rod  from  a  jar  of  95%  alcohol 
we  flame  it  and  to  cool  the  same  we  press  the  bent  portion  into  the  middle  of  the 
plate.  This  also  divides  the  surface  of  the  plate  into  two  portions.  Then  rubbing 
the  bent  rod  over  the  smaller  amount  of  the  material  on  the  right  side  we  carry  it  over 
the  entire  right  side.  Then  go  to  the  loopful  deposited  on  the  left  side  with  the  rod 
and  rub  it  over  this  side.  For  urine,  deposit  i  drop  on  one  side  and  5  drops  on 
the  other.  A  smear  from  pus,  sputum,  urine  or  throat  culture  should  always  be  made 
first  in  order  to  get  an  idea  as  to  the  degree  of  dilution  which  is  necessitated  before 
plating  out.  We  use  blood  agar  plates  as  routine  ones  when  we  culture  from 
throat,  glands,  joints,  pus,  blood,  etc.  The  lack  of  translucency  does  not  interfere 
when  we  study  the  morphology  of  superficial  colonies,  using  a  hand  lens.  Then 
the  haemolytic  zone  of  S.  pyogenes  or  the  green  of  S.  viridans  or  the  Pneumococcus 
make  such  a  medium  indispensable.  All  pathogens  grow  well  on  it.  For  faeces 
we  use  Endo's  medium. 

Esmarch's  Roll  Culture  Tubes. — Having  melted  about  5  c.c.  agar  in  a  test- 
tube  we  inoculate  the  melted  medium  at  45°C.  and  very  quickly  roll  the  tube  in 
a  groove  melted  out  of  a  block  of  ice.  The  agar  sets  on  the  sides  of  the  tube  and 
colonies  may  be  studied  with  a  glass.  Such  tubes  form  a  large  amount  of  water 
of  condensation  which  aids  in  the  study  of  streptococci.  By  inoculating  as  above, 
heating  to  8o°C.,  then  rolling  and  filling  the  tube  with  liquid  petrolatum  we  have 
a  simple  method  for  anaerobes.  Larger  tubes  with  rolled  media  are  useful  for 
culturing  bacterial  growth  for  vaccines,  this  technique  giving  a  larger  surface 
than  the  slant. 

To  obtain  isolated  colonies  on  blood-serum  or  blood-streaked  agar, 
which  can  be  touched  and  by  transfer  obtained  in  pure  culture,  we 
simply  smear  the  material  on  a  slant  of  either  medium.  Then,  without 
sterilizing  the  loop,  we  smear  it  thoroughly  over  a  second  slant,  and  so 
on  to  a  third,  or  possibly  a  fourth  or  fifth. 

Classification. — At  present  the  classification  of  the  bacteria  is  very  unsatisfactory 
from  a  scientific  standpoint.  The  nomenclature  abounds  in  instances  where  three 
or  four  terms  are  used  in  naming  a  single  bacterium,  instead  of  the  single  generic 
name  and  single  specific  one  as  is  used  in  zoological  nomenclature.  This  matter  of 
nomenclature  is  a  subordinate  factor  in  the  confusion  when  we  begin  to  investigate 
and  find  that  different  names  have  been  applied  to  apparently  the  same  organism. 

The  slightest  variation  in  morphological,  locomotor,  or  biological 
characteristics  seems  to  be  considered  sufficient  by  many  observers  to 
justify  the  description  of  a  new  species,  and,  of  course,  the  giving  of  a 
new  name.  Many  of  these  names  which  are  now  retained  were  applied 
prior  to  the  epoch-making  introduction  of  gelatin  media  by  Koch  (1881) 


CLASSIFICATION  51 

and  consequently  at  a  time  when  the  isolation  of  organisms  in  pure 
culture  was  a  matter  of  extreme  difficulty  and  uncertainty.  One  of  the 
first  facts  noted  by  the  student  in  taking  up  bacteriology  is  the  diffi- 
culty in  determining  motility;  this  property  should  always  be  tested 
on  young  cultures  in  bouillon.  In  Brownian  movement  there  is  a  sort 
of  scintillating  movement,  but  the  bacterium  does  not  move  from  that 
part  of  the  field.  In  current  movement  all  the  bacteria  swarm  in  the 
same  direction,  going  very  fast  at  times,  and  then  more  slowly.  If  in 
great  doubt,  the  mounting  of  the  organisms  in  a  2  %  solution  of  carbolic 
acid  will  stop  movement  if  it  be  true  functional  motility,  while  Brownian 
and  current  movement  are  not  interfered  with.  In  true  motility  bac- 
teria move  in  opposite  and  in  all  directions,  and  move  away  from  the 
place  where  first  observed  unless  degenerated  or  dead. 

At  times  we  judge  of  motility  by  the  presence  of  this  characteristic 
in  a  few  of  the  organisms  seen  in  the  microscopic  field,  the  vast  majority 
of  the  bacteria  not  showing  motility.  A  source  of  error  can  be  present 
when  the  bacteria  are  emulsified  in  a  drop  of  water  which  might  contain 
motile  bacteria. 

Reaction  of  media  is  a  factor  of  the  greatest  importance  in  causing  variation  in 
the  functions  of  bacteria,  and  is  one  which  has  until  recently  been  almost  entirely 
neglected.  In  describing  an  organism  at  the  present  time  it  is  always  necessary 
to  note  the  reaction  of  the  media,  the  temperature  at  which  cultivation  took  place, 
and  the  age  of  the  culture  when  examined. 

In  the  following  keys  the  term  bacterium  has  been  used  as  a  general 
designation  for  all  schizomycetes.  Migula  calls  motile  rod-shaped 
organisms  bacilli,  and  nonmotile  ones  bacteria.  Lehmann  and  Neu- 
mann call  spore-bearing  organisms  bacilli,  and  nonspore-bearing  ones 
bacteria. 

The  B.  typhosus  is  very  motile  and  does  not  possess  spores.  According  to  Migula, 
it  would  be  the  Bacillus  typhosus;  according  to  Lehmann  and  Neumann,  the  Bac- 
terium typhosum.  The.  B  anthracis  has  spores  and  is  nonmotile.  Hence  it  would  be 
Bacterium  anthracis,  according  to  Migula,  and  Bacillus  anthracis,  according  to 
Lehmann  and  Neumann. 

In  the  use  of  the  keys  at  the  head  of  each  group  of  organisms  it  will  be  observed 
that  the  primary  separation  is  on  the  basis  of  morphology — the  cocci  in  one  group, 
the  bacilli  in  three  subgroups:  one  for  those  rod-shaped  organisms  showing  branch- 
ing and  curving  forms,  one  for  the  spore  bearers  and  one  for  the  simple  rods.  The 
spirilla  are  grouped  by  themselves. 

An  important  method  of  differentiation  is  the  reaction  to  Gram's 
stain.  It  should  be  remembered  that  organisms  carried  along  on  arti- 


STUDY  AND   IDENTIFICATION   OF  BACTERIA 


CHART  FOR  STUDY  OF  BACTERIA 


.Date. 


Name JSource 

Tbrm Arrangement. . 

Size,  length Mreadih Extreme  length.... 

Capsules Spores, Central Terminal. 

Motility. Pleomorphism .._;._ 

Staining,  reaction,  Loffler, Gram, CLcid  fast. 

Pathogenesis-.-   White  mouse Guinea  pig. J?abbii. 

AEROBIC     OR       FACULTATIVE 


N.RGlucose  L.Mannite         L.Dulcite          L Maltose       L.Saccharose   Lit.Lactose 


Gelatin   stab 


A 


Qgar  slant 


Cigar, 
Glycerin    agar 

OBLIGATE    ANAEROBIC 


Stood    agar,or  Endo-or 

Serum      agar  Lactose  litmuJ3 


tyar 


A 


Glucose  agar          Litmus    milk  Egg  media,  or  Sterile   tissut 

Biocd    serum,  Glucose  bouillon 


stab 


Special   media: 

Notes-.... 


FIG.  io.— Bateriological  Chart  in  use  at  the  U.  S.  Naval  Medical  School.  The 
mimeographed  sheets  are  8  by  14  inches.  Red  and  blue  pencil  shading  charac- 
terizes acid  or  alkaline  reactions  in  sugar  tubes.  Outlines  of  colony  in  plate  rings 
are  made  in  pencil  as  is  also  done  on  slant  figures. 


MOTILITY 


53 


ficial  media  often  lose  their  Gram-staining  characteristics;  hence  it  is 
desirable  to  determine  this  staining  reaction  in  cultures  freshly  isolated. 
Be  sure  that  the  stains,  especially  the  aniline  gentian  violet  and  the 
iodine  solution,  have  not  deteriorated.  There  is  no  more  important 
stain  than  this,  and  none  which  requires  greater  experience.  The  chief 
causes  of  conflicting  results  are  i.  working  with  old  cultures  and  2.  not. 
having  satisfactory  staining  solutions. 


FIG.  ii.— Series  of  stab  cultures  in  gelatin,  showing  modes  of  growth  of  different 
species  of  bacteria.     (Abbott.') 

Motility,  as  stated  above,  is  at  times  difficult  to  determine.  For  this  purpose 
young  eighteen-hour-old  bouillon  cultures  are  preferable,  and  the  preparation  should 
be  made  by  applying  a  vaseline  ring  to  the  slide,  then  putting  a  drop  of  the  bouillon 
culture  in  the  center  of  the  ring  (or  a  drop  of  water  inoculated  from  an  agar  slant 
growth),  then  putting  on  a  cover-glass.  By  this  method  current  movement  is  done 
away  with  and  the  preparation  keeps  for  hours.  This  is  a  convenient  method  for 
agglutination  tests.  The  hanging  drop  with  a  concave  slide  is  ordinarily  used. 
With  this,  cut  down  the  light  and  focus  on  the  margin  of  the  drop  with  the  %- 
inch  objective  before  examining  with  a  high  dry  objective  (%  inch). 


54  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

Liquefaction  of  gelatin  is  a  very  important  means  of  differentiating.  When  room- 
temperature  incubator  is  not  at  hand  (20°  to  22°C.),  it  is  better  to  put  the  inocu- 
lated gelatin  tube  in  the  body-temperature  incubator,  and  from  day  to  day  test  the 
power  of  solidifying  with  ice- water.  If  the  organism  digests  the  gelatin  (a  liquefier), 
the  medium  will  remain  fluid  when  placed  in  ice- water;  if  the  organism  is  a  non- 
liquefier,  the  medium  in  the  tube  becomes  solid.  Of  course  we  lose  the  information 
to  be  obtained  from  the  shape  of  the  area  of  liquefaction. 

For  routine  work  the  only  sugar  media  used  are  glucose  and  lactose 
bouillon.  These  are  of  the  utmost  importance  in  differentiating  organ- 
isms of  the  typhoid  and  colon  group.  Following  Ford,  these  intestinal 
bacteria  have  primarily  been  separated  by  their  action  on  litmus  milk 
— whether  turning  it  pink  or  only  slightly  changing  or  not  changing  at 
all  the  original  color. 

Colony  Isolation. — Examine  the  colonies  on  Petri  plate  at  first  with  the  unaided 
eye,  then  with  a  hand  magnifying  glass  or  low-power  objective,  using  reflected  and 
transmitted  light  alternately.  Having  determined  the  presence  of  two  or  more  dif- 
ferent kinds  of  colonies,  make  a  ring  with  wax  pencil  around  one  or  more  of  each 
kind  of  colony,  numbering  them.  The  slides  or  culture  tubes  used  in  determining 
the  species  of  organism  present  in  the  plate  should  bear  the  same  number  as  that 
of  the  colony  from  which  the  material  was  taken.  A  convenient  procedure  is  to 
put  a  loopful  of  water  on  a  clean  coverglass  and  emulsify  material  from  a  colony 
in  it.  Then  invert  over  a  concave  slide  without  vaselining  the  circumference  of 
the  concavity.  After  examining  for  motility,  smear  out  and  dry  the  bacterial 
preparation.  Then  fix  in  the  flame  and  stain  with  aniline  gentian  violet  for  two 
to  five  minutes.  Wash  and  mount  the  preparation  in  water.  Afterward  pass 
through  the  usual  Gram  technic. 

After  this  inoculate  the  various  culture  media  from  similar  colonies.  One  may 
inoculate  a  tube  of  bouillon  from  a  single  colony  and  later  on  inoculate  the  other  culture 
tubes. 

In  testing  for  gas  production  it  is  better  to  use  the  Durham  fermenta- 
tion tube  as  small  amounts  of  gas  may  not  be  easily  detected  with  deep 
stab  cultures  into  glucose  or  lactose  agar. 

If  a  Durham  or  Smith  tube,  or  a  slant  of  Russell's  double  sugar  medium  be  not  at 
hand  the  production  of  gas  may  be  determined  by  observing  bubble  formation  on  the 
surface  of  the  sugar  bouillon  culture.  As  none  of  the  pathogenic  cocci  produce  gas, 
fermentation  tubes  are  unnecessary  where  cocci  are  to  be  studied.  The  litmus  milk 
tube  gives  data  as  to  acid  production. 

An  important  point  is  to  wait  at  least  forty-eight  hours  (in  the  case 
of  M .  melitensis,  four  to  seven  days)  before  reporting  on  the  cultural 
findings  on  the  agar,  blood  agar  or  blood-serum  slant  or  plate  upon 
which  the  material  is  smeared  (pus,  exudate,  blood,  etc.). 

Indol  production  is  of  but  slight  aid  in  differentiating  organisms. 


55 

Anaerobiasis. — If  it  were  not  for  the  fact  that  we  have  so  many  facultative  anaerobes 
(organisms  growing  under  anaerobic  as  well  as  aerobic  conditions)  it  would  be  of 
practical  utility  to  make  this  biological  variation  our  first  step  in  the  study  of  an 
unidentified  organism.  At  any  rate  it  is  well  to  remember  that  the  causative  organ- 
isms of  plajue,  tuberculosisfmfluenza,  gonorrjioea.  pneumococcal  pneumonia  and 
glanders  are  obligate  aerobes  ^/vhile  those  oTftetanus,  botulism,  gasjfangrene  and 
malignant  oedema  are  obligateanaerobesj  The  p^ogenic  cocci  as  well  as  the  causa- 
tive organisms  of  choleraTtyprioid,  parathyphoid  and  anthrax  are  facultative  anae- 
robes; they  are,  however,  always  studied  under  aerobic  conditions.  The  colon 
bacillus  as  well  as  organisms  of  the  Friedlander  group  are  also  facultative  anaerobes. 

Should  an  organism  be  encountered  in  original  investigations  these 
requirements  as  to  etiological  relationship  should  be  carried  out  (Koch's 
postulates) : 

i.  The  organism  should  be  constantly  present  in  that  particular  pathological 
condition.  2.  Such  bacteria  should  be  isolated  in  pure  culture  from  the  pathologi- 
cal material.  3.  Such  pure  cultures  when  inoculated  into  suitable  animals  should 
reproduce  the  pathological  conditions  and  should  be  capable  of  a  second  isolation  in 
pure  culture  from  such  an  experimental  animal.  For  various  reasons,  such  as  unsuit- 
able animals  or  artificial  media,  these  requirements  are  impossible  of  execution  with 
several  organisms  which  are  generally  recognized  as  the  causes  of  certain  diseases. 

ANIMAL  EXPERIMENTATION 

The  experimental  animals  most  frequently  employed  in  the  diagno- 
sis of  bacterial  diseases  are  the  guinea-pig,  the  rabbit,  the  white  rat 
and  the  white  mouse.  In  the  following  diseases  the  most  suitable  ani- 
mals for  inoculation  are: 

1.  Tetanus — mice  or  guinea-pigs,  subcutaneously.    The  spasms  begin  in  the 
limbs  nearest  the  site  of  inoculation. 

2.  Pneumococci  and  streptococci — mice,  intraperitoneally,  or  rabbits,  intraven- 
ously. 

3.  Staphylococci — rabbits. 

4.  Diphtheria,    tuberculosis,    anthrax   and   malignant  cedema— the  guinea-pig, 
subcutaneously. 

5.  Glanders  and  cholera— the  guinea-pig,  intraperitoneally. 

6.  Plague— guinea-pigs,  cutaneously  or  subcutaneously. 

In  the  cutaneous  method  of  infection  the  material,  as  from  a  plague  bubo,  or  the 
sputum  from  pneumonic  plague,  is  thoroughly  rubbed  with  a  glass  rod  upon  the 
shaven  surface  of  the  guinea-pig. 

In  the  subcutaneous  method  one  can  use  a  hypodermic  needle  (the  all-glass 
syringe  with  platino-iridium  needle  is  the  best)  or  an  opening  can  be  cut  with  the 
scissors,  a  pocket  then  opened  up  with  the  forceps  and  a  piece  of  tissue  inserted  to 
the  bottom  of  the  pocket  with  the  forceps. 

One  can  seal  the  incision  with  collodion. 


56  STUDY  AND   IDENTIFICATION  OF  BACTERIA 

The  large  ear  vein  of  the  rabbit  is  used  for  intravenous  inoculation.  This  can  be 
made  to  stand  out  with  either  hot  water  or  xylol. 

In  intraperitoneal  injections  the  animal  is  best  held  head  down  so  that  the  intes- 
tines gravitate  downward.  The  shaven  skin  is  pinched  up  and  the  needle  inserted  in 
the  median  line. 

In  certain  cases  infection  is  secured  by  feeding  the  material  to  the  experimental 
animal.  The  material  may  be  mixed  with  the  food  or  introduced  into  the  stomach 
by  a  tube. 


CULIURING  MATERIAL  OBTAINED  AT  BIOPSY  OR  AUTOPSY  OF  MAN  OR 

ANIMALS 

It  is  customary  to  sear  with  a  heated  knife  or  spatula  a  spot  on  the 
surface  of  the  organ  from  which  cultures  are  to  be  made.  Then  intro- 
duce at  this  point  a  sterile  platinum  spud  and  inoculate  tubes  of  culture 
media.  The  platinum  loop  may  be  used  where  an  incision  is  made 
into  the  organ  with  a  sterile  knife.  The  ordinary  platinum  loop,  how- 
ever, bends  too  easily. 

A  bacteriological  capillary  pipette  is  a  good  instrument  for  taking  up  material. 
After  some  practice  one  can  do  good  work  with  a  rubber  bulb  capillary  pipette, 
especially  in  taking  up  blood  from  right  heart  or  blood-vessels.  When  great  pre- 
caution is  necessary,  as  in  culturing  a  removed  gland  or  organ,  the  piece  of  tissue 
may  be  dropped  into  5%  formalin  solution  for  a  few  minutes,  then  washed  in  sterile 
salt  solution;  next  placed  in  a  sterile  Petri  dish  and  the  material  obtained  from  the 
center.  It  may  be  dropped  for  a  few  seconds  into  boiling  water  to  sterilize  the 
surface.  When  autopsying  experimental  animals  it  is  well  to  dip  the  dead  animal 
into  3%  tricresol  solution  before  opening  up  the  body. 


CHAPTER  V 

STUDY  AND  IDENTIFICATION  OF  BACTERIA— COCCI.    KEY 

AND  NOTES 

Streptococcus  Forms. — Cells  divide  to  form  chains. 
I.  Gelatin  not  liquefied. 

1.  Haemolytic  zone  on  blood  agar. 

(a)  Very  slight  acidity  in  lactose  litmus  bouillon.  S.  pyogenes.  Tends  to 
produce  arthritis  in  experimental  animals.  Often  a  granular  sediment  in 
bouillon. 

(&)  Marked  acidity  but  no  gas  production  in  jactose  litmus  bouillon.  S. 
lacticus.  Nonpathogenic.  Forms  diffuse  cloudiness  in  bouillon. 

2.  G<re^nish  appearance  about  colonies  on  blood  agar. 

(a)  No  tendency  to  capsule  formation.     S.  viridans.    Produces  endocarditis 
*•  •         in  experimental  animals. 

(&)  Distinct  capsule  formation  in  pathological  material  or  on  favorable  media. 
S.  lanceolatus  (Pneumococcus).  Gram-positive,  lance-shaped  cocci  with 
bases  apposed  within  a  capsule. 

(c)  Very  marked  capsule  development  on  all  media.    S.  mucosus.     A.  strepto- 
coccus with  extraordinary  capsule  development,  up  to  loju  in  width,  S. 
mesenterioides,  is  not  pathogenic. 
II.  Gelatin  liquefied. 

Streptococcus  coli  gracilis.     (Cocci  quite  small — 0.2  to  0.4/1.     In  faeces.) 

A  tube-like    liquefaction;    chains    rather  long;    only  slight  growth  on  agar. 

Constant  inhabitant  of  stools  of  meat  diet. 
Sarcina  Forms. — Cells  divide  in  three  dimensions  of  space.     (Packets). 

A.  No  pigment  production  on  agar. 

(a)  Sarcina  alba.     (Colonies  finely  granular.) 

(b)  Sarcina  pulmonum. 

B.  Yellowish  pigment. 

(a)  Sarcina  lutea.     (Colonies  coarsely  granular.) 
(b)\ kSarcina  flava.     (Colonies  finely  granular.) 

C.  Rose-red  pigment. 
(a)  Sarcina  rosea. 

Micrococcus  Forms. — Cells  divide  irregularly  in  various  directions. 
I.  Gram-positive  cocci. 
A.  Cocci-round. 

i.  Divide  in  two  planes  at  right  angles.    Tetrad  formation.     Merismopedia. 
(a)  M.  tetragenus.     Mo'ist  white  viscid  colonies.     No  liquefaction  of  gela- 
tin.    Capsule. 

57 


58  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

2.  Divide  irregularly.     Bunch  of  grapes  arrangement.     (Staphylococci.) 
(fl)  Gelatin  not  liquefied.     M.  cereus  albus. 

(6)  Gelatin  liquefied.      (  "'  (Staphytococcu.)  pyogenes  albus. 

(  M.  (Staphylococcus)  pyogenes  aureus. 
(c)   Gelatin  very  slightly  liquefied. 

S.  epidermidis  albus.     (Stitch  coccus.) 
B.  Cocci — biscuit-shape. 

Diplococcus  crassus.     (May  be  mistaken  for  Meningococcus.) 
On  ordinary  agar  we  have  a  scanty  growth  resembling  the  Streptococcus.     Colo- 
nies on  ascites  agar  are  smaller  than  those  of  Meningococcus.     It  produces  acid  in 
glucose,  maltose,  lactose  and  saccharose. 
II.  Gram-negative  cocci. 

only  at  about  incubator  temperature. 

1.  Grow  only  on  blood  or  serum  media.     Gonococcus. 

2.  Grow  on  blood-serum  media,  or  glycerine  agar. 

(a)  Diplococcus  intracellularis_rneningitidis.     Produces  acid  in  glucose 

and  maltose  but  not  in  lactose  nor  saccharose. 
\^.  Grows  on  ordinary  media.     Micrococcusjnelitensis. 
B.  will  grow  at  room  temperature  as  well  as  at  37°C. 

(a)  Micrococcus  catarrhalis.     Does  noj:  produce  acid  in  glucose,  maltose, 
lactose  nor  saccharose.  ^ 

(b)  M.  pharyngis  siccus.     Colonies  dry  and  tough  and  adhere  to  medium. 
NOTE. — Other  biscuit-shaped  Gram-negative  organisms  resembling  the  Meningo- 
coccus are  (a)  Diplococcus  flavus.     The  colonies  show  yellow  pigment  and  we  have 
three  varieties  according  to  the  depth  of  the  yellow  color,     (b}  M.  pharyngis  siccus, 
with  yellowish,  dry  tenacious  colonies  and  (c)  M.  cinereus  with  coarse  dry  colonies 
on  ascitic  agar.    Like  M .  catarrhalis,  it  does  not  ferment  any  of  the  above-mentioned 
sugars.     The  individual  cocci,  however,  are  larger  and  more  oblong  in  shape. 

STREPTOCOCCUS  FORMS 

Those  cocci  tending  to  arrange  themselves  in  chains  are  usually 
described  as  streptococci.  (Ogston,  1881;  Rosenbach,  1884.) 

When  we  consider  that  certain  bacilli  at  times  assume  an  arrange- 
ment which  we  term  streptobacilli,  yet  have  no  relationship,  it  would 
suggest  that  the  matter  of  chain  morphology  is  simply  a  characteristic 
common  to  many  entirely  different  cocci. 

Again  old  laboratory  cultures  of  streptococci  may  show  alternations  of  cocci  and 
rods  giving  the  appearance  of  the  dots  and  dashes  of  the  Morse  code.  Furthermore 
unsuitable  media  may  bring  about  various  involution  types  in  an  organism  pri- 
marily streptococcal. 

It  is  often  difficult  to  distinguish  streptobacilli  from  streptococci  morphologically 
and  the  same  is  true  of  diplococci  and  diplobacilli.  These  bacillary  pairs  and  chains, 
however,  often  show  bipolar  staining  and  are  almost  invariably  Gram-negative. 

While  streptococci  tend  to  assume  chain  formation  in  pus  and  tissues  they  often 
appear  as  diplococci  in  blood. 


VIRULENCE  IN  STREPTOCOCCI  59 

The  essential  point  to  bear  in  mind  is  that  the  finding  of  a  strepto- 
coccus does  not  necessarily  explain  an  infection,  because  normally 
streptococci  are  among  the  organisms  most  frequently  and  abundantly 
found  in  plates  made  from  normal  buccal  and  nasal  secretions.  It  is 
well  to  be  very  conservative  When  reporting  streptococci  as  the  etiolog- 
ical  factor  from  lesions  of  the  throat  or  nose. 

Probably  the  most  practical  point  in  the  differentiation  of  streptococci,  next  to 
that  of  pathogenicity,  is  the  occurrence  of  long  or  short  chains,  the  virulent  ones 
tending  to  appear  in  chains  of  from  10  to  20  cocci,  while  the  normal  inhabitants  of 
the  nose,  mouth  and  faeces  generally  tend  to  be  in  shorter  chains. 

As  regards  virulence,  this  is  exceedingly  variable — it  is  soon  lost,  but 
may  be  restored  either  by  inoculating  streptococci  along  with  various 


FIG.  12. — Streptococcus  pyo genes,     (Kolle  and  W  assermann.) 

other  organisms  or  by  passage  through  successive  rabbits.  The  rabbit 
is  the  most  susceptible  animal  and  should  be  inoculated  in  one  of  the 
prominent  ear  veins.  If  the  needle  of  the  syringe  is  not  inserted  in 
the  vein  it  will  be  difficult  to  force  in  the  material  and  a  swelling  will 
immediately  show  itself. 

Recently  isolated  cultures  from  human  infections  are  not  very  virulent  for  animals. 
Passage  through  the  rabbit,  however,  enormously  increases  the  virulence.  It  may 
be  more  convenient  to  inoculate  a  mouse  at  the  root  of  the  tail.  If  the  culture  is 
very  virulent,  it  becomes  generalized  and  death  occurs  in  two  or  three  days.  If  less 
virulent,  a  local  abscess  forms. 

Besides  the  morphological  and  pathogenic  variations,  Schottmuller 
has  noted  differences  where  these  organisms  are  grown  on  i  part  of 


60  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

blood  and  3  to  6  parts  of  agar.  On  this  medium  Strep,  erysipdatis  has 
a  hemolytic  action,  the  laking  of  the  red  cells  bringing  about  a  more 
or  less  clear  ring  surrounding  the  colony.  This  organism  is  often  termed 
S.  hcemolyticus.  It  tends  to  produce  suppurative  arthritis  in  rabbits 
while  S.  viridans  causes  endocarditis  and  the  Pneumococcus  an  acute 
sepsis. 

The  haemolytic  substance  is  much  like  a  ferment  and  is  found  in  filtrates  of  cul- 
tures. The  short-chain  streptococci  do  not  have  a  haemolytic  halo.  They  also 
have  a  greenish  appearance  like  the  Pneumococcus  (S.  viridans}.  The  Pneumococcus 
has  a  greenish  zone.  Streptococci  which  are  profoundly  toxic  and  which  have  been 
isolated  from  milk-borne  epidemic  sore  throats  differ  from  the  ordinary  S.  pyogenes 
in  being  encapsulated,  not  tending  to  form  chains  and  producing  only  slight  haemoly- 
sis on  blood  agar. 

Some  of  the  English  authorities  have  introduced  biochemical  meth- 
ods of  differentiating:  the  Strep,  pyogenes  coagulating  milk,  reducing 
neutral  red,  and  producing  acid  in  lactose,  saccharose  and  mannite 
media. 

S.  pyogenes  does  not  produce  acid  in  inulin  media  while  the  Pneumococcus  does. 

The  Pneumococcus  and  Streptococcus  mucosus  ferment  inulin  and  give  greenish 
colonies.  Of  streptococci  not  fermenting  inulin  we  have  S.  pyogenes,  with  its  haemo- 
lytic  zone  around  the  colony;  S.  viridans,  with  its  green  colony,  and  S.  f&calis,  which 
gives  colonies  neither  distinctly  green  nor  haemolytic. 

A  freshly  prepared  solution  of  sodium  taurocholate,  5%,  added  to  an  equal 
amount  of  a  twenty-four-hour  bouillon  culture  of  S.  pyogenes  does  not  disintegrate 
the  cocci  or,  at  any  rate,  not  within  a  few  minutes.  The  reverse  is  true  of  the 
Pneumococcus. 

(  iWhen  we  consider  the  biochemical  variations  which  a  single  organism,  as  the 
colon  bacillus,  may  exhibit,  the  value  of  such  methods  of  differentiating  may  well  be 
questioned.  The  question  of  the  symbiotic  relationship,  which,  when  established 
between  two  or  more  bacteria,  may  cause  harmless  organisms  to  take  on  virulence, 
would  appear  to  be  a  more  important  consideration. 

Almost  without  exception,  human  streptococci  are  Gram-positive. 
Their  colonies  are  quite  small  but  distinct  and  discrete.  In^appear- 
ance  the  colonies  of  streptococci  and  pneumococci  are  practically  iden- 
tical. In  a  blood-serum  throat  culture  Pneumococcus  and  Streptococcus 
colonies  are  the  smallest,  diphtheria  ones  are  quite  small  and  discrete, 
but  slightly  flatter.  (Always  examine  the  water  of  condensation  for 
streptococci.)  The  Sarcina  and  Staphylococcus  colonies  are  much 
larger. 

Streptococcic  colonies  on  blood  agar  are  much  more  moist  and  luxuriant  than  on 
ordinary  agar.  A  very  important  point,  in  judging  whether  a  Streptococcus  or 


GASTRIC  ULCER  AND  STREPTOCOCCI  6 1 

other  organism  is  pathogenic  in  a  given  infection,  is  to  examine  smears  from  the  pus 
or  other  material  in  a  Gram-stained  specimen  for  information  as  to  abundance  and, 
in  particular,  phagocytosis  of  any  organism,  before  plating  out. 

Streptococci  are  commonly  the  cause  of  diffuse  phlegmonous  inflam- 
mations, while  the  staphylococci  cause  circumscribed  lesions.  Strepto- 
cocci cause  necrosis  and  do  not  characteristically  produce  pus.  The 
importance  of  the  Streptococcus  as  a  secondary  infection  in  diphtheria, 
tuberculosis,  small-pox,  and  even  in  typhoid  fever  must  always  be  kept 
in  mind.  It  is  this  infection  which  does  not  respond  to  diphtheria 
antitoxin,  and  not  the  diphtheria  one. 

Rosenow  has  reported  on  the  rather  constant  presence  of  streptococci  in  gastric 
and  duodenal  ulcers  removed  at  operation,  under  which  circumstance  the  number  and 
variety  of  bacteria  present  are  comparatively  few.  The  strains  from  27  chronic 
ulcers  gave  grayish-green  colonies  on  blood  plate,  were  in  short  chains  and  diplococci, 
produced  much  acid  and  turbidity  in  dextrose  broth  and  showed  a  low-grade  virulence. 
When  injected  into  dogs,  rabbits  and  guinea-pigs  they  showed  a  tendency  to  localize 
in  the  mucosa  of  stomach  and  duodenum,  causing  ulceration  in  a  large  percentage 
of  cases. 

Streptococci  as  well  as  colon  infections  are  always  to  be  thought  of  in  connection 
with  cholecystitis  and  appendicitis. 

It  has  been  claimed  that  scarlet  fever  is  a  streptococcal  infection  (S.  anginosus). 
Klimenko  found  streptococci  only  n  times  in  the  blood  of  523  cases  of  scarlet 
fever.  The  Dohle  inclusion  bodies  of  the  disease  suggest  chlamydozoal  virus. 
Mallory  has  very  recently  claimed  that  scarlet  fever  is  due  to  a  diphtheria-like 
bacillus.  It  is  found  in  the  same  locations  as  the  diphtheria  organism  and  also 
produces  a  toxin  which,  however,  is  less  virulent  and  only  produces  inconspicuous 
lesions.  The  membrane  formation  in  the  throat  in  scarlet  fever  is  due  to  streptococci. 

When  freshly  isolated  from  human  lesions  streptococci  often  show  only  a  slight 
virulence  for  animals.  Hence  massive  doses  are  indicated  and  intravenous  or  intra- 
peritoneal  injections.  The  guinea-pig  is  not  very  susceptible  to  streptococci;  the 
rabbit  and  white  mouse  being  the  animals  of  choice. 

In  nondiphtheritic  anginas,  puerperal  fever,  ulcerative  endocarditis 
and  coccal  enteritis  it  is  the  Streptococcus  which  is  usually  the  cause. 
It  has  been  claimed  that  acute  articular  rheumatism  is  due  to  a  short- 
chain  streptococcus  (M.  rheumaticus),  which  is  best  isolated  from 
material  from  an  acute  joint  infection,  but  may  also  be  isolated  occa- 
sionally from  the  blood.  It  produces  much  acid  and  clots  milk  in  two 
days.  The  growth  is  described  as  being  more  luxuriant  than  that  of 
S.  pyogenes.  It  is  about  0.5^  in  diameter. 

The  majority  of  investigators  have  reported  streptococci  from  acute  joint  inflam- 
mations and  bacilli  from  chronic  infectious  joint  affections.  Goadby  has  considered 
a  streptobacillus,  somewhat  resembling  Ducrey's  bacillus  of  chancroid,  which  ex- 


62  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

hibits  marked  pleomorphism  and  Gram  variations,  and  grows  best  on  egg  albumin 
agar  of  plus  3  reaction  as  the  cause  of  arthritis  deformans  and  alveolar  osteitis. 
Inoculation  of  cultures  of  this  organism  into  or  around  the  knee-joints  of  rabbits 
has  produced  lesions  similar  to  those  of  rheumatoid  arthritis. 

Of  the  nonpathogenic  streptococci  the  most  important  one  is  S.  lacticus,  which 
is  described  under  milk.  This  differs  from  S.  pyogenes  in  growing  at  lower  tempera- 
tures and  having  greater  viability.  It  is  a  normal  inhabitant  of  cows'  dung. 


SARCINA  FORMS 

These  are  best  observed  in  hanging-drop  preparations,  when  they 
can  be  seen  as  little  cubes,  like  a  parcel  tied  with  a  string,  and  by  noting 
them  when  turning  over,  it  will  be  seen  that  they  are  different  from  the 
tetrads  which  only  divide  in  two  directions  of  space.  At  times  the 
packet  formation  is  not  perfect  and  it  will  be  difficult  to  distinguish 
such  as  sarcinae.  All  sarcinae  stain  by  Gram.  If  the  staining  of  sar- 
cinae  be  too  deep  it  may  obscure  the  lines  of  cleavage.  Sarcinae  are  non- 
motile. 

i  ^Various  sarcinae  have  been  isolated  from  the  stomach,  especially  when  there  is 
stagnation  of  stomach  contents.  Sarcinse  have  also  been  found  in  the  intestines. 
In  plates  the  S.  lutea  is  frequently  a  contaminating  organism,  being  rather  constantly 
present  in  the  air.  The  demonstration  of  sarcina  morphology  should  always  be 
made  from  liquid  media,  as  bouillon.  Urine  makes  an  excellent  medium. 

MICROCOCCUS  FORMS 

This  grouping  includes  all  cocci  which  do  not  show  chain  or  packet 
formation.  It  will  be  found  convenient  to  divide  them  into  two  classes 
according  to  their  staining  by  Gram.  The  M.  tetragenus,  S.  pyogenes 
aureus  and  the  Pneumococcus  stain  by  Gram,  while  the  Gonococcus, 
the  Meningococcus,  the  M .  catarrhalis  and  the  M .  melitensis  are  Gram- 
negative. 

M.  Tetragenus. — This  organism  is  frequently  found  associated  with 
other  organisms  in  sputum,  especially  with  tubercle  and  influenza 
bacilli.  The  colonies  are  white,  slightly  smaller  than  staphylococci  and 
are  quite  viscid. 

It  was  formerly  considered  unimportant  in  disease,  but  the  idea  now  prevails  that 
it  is  responsible  for  many  abscesses  about  the  mouth,  especially  in  connection  with 
the  teeth.  Injected  subcutaneously  into  Japanese  mice,  it  produces  a  septicaemia 
and  death  in  three  or  four  days.  The  blood  shows  great  numbers  of  encapsulated 
tetrads.  It  has  been  reported  twice  as  a  cause  of  septicaemia  in  man. 


STAPHYLOCOCCI 


Staphylococci. — To  cocci  dividing  irregularly  and  usually  forming 
masses  which  are  likened  to  clusters  of  grapes  the  term  Staphylococcus 
is  applied.  While  there  have  been  experiments  which  show  that  by 
selecting  pale  portions  of  a  yellow  colony,  eventually  a  white  colony 
could  be  produced,  yet,  as  a  practical  consideration,  it  is  convenient 
to  consider  at  least  two  types  of  Staphylococci:  the  Staphylococcus  pyo- 
genes aureus  and  the  Staphylococcus  pyogenes  albus. 
In  culturing  from  the  pus  of  an  abscess  or  furuncle 
we  generally  obtain  a  golden  coccus,  while  in  ma- 
terial from  the  nose  or  mouth,  the  Staphylococcus 
colonies  are  almost  invariably  white.  As  regards 
the  common  skin  coccus,  this  will  be  found  to  pro- 
duce a  white  colony.  A  coccus  which  very  slowly 
liquefies  gelatin  and  has  been  supposed  to  cause 
stitch  abscesses  is  the  6*.  epidermidis  albus. 

While  it  is  customary  to  look  for  a  golden  colony  in  the 
case  of  organisms  showing  virulence,  yet  at  times  a  cream- 
white  colony  may  develop  from  cocci  of  great  virulence. 
Staphylococci  show  marked  resistance  to  dessication  and 
dried  pus  may  contain  live  organisms  for  months.  Old 
bouillon  cultures  of  Staphylococci  contain  a  ferment-like 
substance,  leucocidin,  which  disintegrates  leukocytes.  Such 
cultures  may  also  show  a  haemolysin  and  when  filtered  and 
injected  into  animals  show  destructive  action  on  cells  of 
various  organs.  Amyloid  change  may  be  caused  in  animals 
by  repeated  injections  of  either  living  or  dead  cultures. 

The  S.  pyogenes  citreus  is  considered  as  of  very  feeble 
pathogenic  power.     Certain  cocci  whose  colonies  have  pre- 
sented   a    waxy  appearance  have  been  designated  as  S. 
cereus  albus  and  S.  cereus  flavus,  respectively.     They  are  of 
very  little  practical  importance.    The  Staphylococcus  pyogenes   JjJJJJ?  ^/teeT  old! 
aureus  grows  readily  at  room  temperature,  but  better  at  37°C.    (Mac  Neal.) 
It  coagulates  milk  and  renders  bouillon  uniformly  turbid. 

It  grows  on  all  media,  as  blood-serum,  agar,  potato,  etc.  It  has  been  proposed  to 
distinguish  it  from  skin  Staphylococci  by  its  power  of  producing  acid  in  mannite. 
Ordinarily  the  individual  cocci  are  about  IJJL  in  diameter,  but  they  vary  greatly  in 
size  according  to  the  age  of  the  culture  and  other  conditions.  The  "aureus/'  as  it 
is  frequently  called,  is  not  only  often  found  in  circumscribed  processes,  but  it  is  a 
frequent  cause  of  septicaemia,  osteomyelitis,  endocarditis,  etc.  In  the  tropics 
staphylococcal  infections  often  show  great  virulence  and  clinically  may  resemble 
streptococcal  ones.  Smears  from  such  erysipelatoid  lesions  show  diplococcal 
morphology,  often  phagocytized.  A  pemphigoid  eruption  in  children  is  often 
staphylococcal  (Pyosis). 

In  infection  of  bone  tissue  the  Staphylococcus  is  by  far  the  most  frequent  cause. 


FIG.    13. — Gelatine 


04  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

It  is  well  to  remember  that  insignificant  staphylococcal  infection  may  lead  to  sep- 
ticaemia. In  the  tropics,  where  resistance  is  often  lowered  and  staphylococcal  skin 
infections  common,  continued  fevers  are  often  septicaemias.  It  is  the  organism 
most  frequently  concerned  in  terminal  infections.  The  lowered  resistance  of  the 
patient  permits  of  their  passage  through  barriers  ordinarily  resistant.  Not  only 
should  this  be  kept  in  mind  when  such  organisms  are  isolated  at  an  autopsy,  but 
as  well  the  fact  that  their  entrance  may  have  been  agonal  or  subsequent  to  death. 
Vaccines  have  been  most  successful  in  the  treatment  of  staphylococcal  infections. 
They  stimulate  phagocytosis.  Pyelitis  may  be  due  to  staphylococci. 

The  Pneumococcus  of  Fraenkel. — (Pasteur  and  Sternberg  in  1880. 
Fraenkel,  1884,  isolated  it  from  normal  persons  as  well  as  pneumonia 
patients.  Inoculated  mice  and  rabbits.  Hence  FraenkePs  organism. 


FIG.  14. — Pneumococcus,  showing  capsule,  from  pleuritic  fluid  of  infected  rabbit, 
stained  by  second  method  of  Hiss.     (Mac  Neal.} 

Weichselbaum  accurately  differentiated  organisms  causing  pneumonia 
in  1886.)  This  is  by  far  the  most  common  cause  of  pneumonia,  whether 
it  be  of  the  croupous,  catarrhal,  or  septic  type.  It  is  also  frequently 
found  in  meningitis,  empyema,  endocarditis  and  otitis  media.  It 
should  not  be  confused  with  the  pneumobacillus  of  Friedlander,  which, 
although  possessing  a  capsule  like  the  Pneumococcus,  differs  from  it  by 
being  Gram-negative,  being  a  bacillus  and  having  large  viscid  colonies. 
The  Pneumococcus  is  the  cause  of  more  than  80%  of  the  cases  of 
pneumonia. 

It  does  not  grow  below  2o°C.  and  is  best  cultivated  on  blood-serum,  or  blood- 
streaked  agar.     On  plain  agar  it  grows  as  a  very  small  dew-drop-like  colony,  which 


THE  PNEUMOCOCCUS  65 

is  slightly  grayish  by  reflected  light.  It  produces  considerable  acid,  thus  acidify- 
ing and  usually  coagulating  litmus  milk.  It  produces  acid  inynulin  media  which 
the  Streptococcus  fails  to  do.  The  colony  is  smaller  and  more  transparent  than  a 
Streptococcus  colony.  In  sputum  or  other  pathological  material  it  is  best  recognized 
by  the  presence  of  a  capsule  inclosed  in  which  are  two  lance-shaped  cocci  with  their 
bases  apposed.  In  artificial  culture  we  rarely  get  the  capsule.  It  also  sometimes 
grows  in  short  chains  like  a  Streptococcus.  The  best  medium  for  differentiating  is 
the  serum  of  a  young  rabbit;  in  this  it  grows  as  a  diplococcus,  while  streptococci 
show  chains.  Kosenow  has  by  combining  passage  in  animals  with  culturing  sym- 
biotically  with  B.  subtilis  changed  the  Pneumococcus  into  a  haemolytic  Streptococcus. 
The  best  method  of  isolating  it  in  pure  culture  is  to  inject  the  sputum  into  the 
marginal  ear  vein  of  a  rabbit  or  subcutaneously  into  a  mouse.  Death  results  from 
septicaemia  in  about  two  days  and  the  blood  teems  with  pneumococci.  Usually 
the  Pneumococcus  quickly  loses  its  virulence,  and  also  dies  out  in  a  few  days  unless 
transferred  to  fresh  media.  The  best  medium  for  its  preservation  is  rabbit's  blood 
agar;  this  also  maintains  the  virulence.  On  this  medium  the  colonies  are  larger 
than  on  agar  and  they  present  a  greenish  appearance. 

The  Pneumococcus  growth  emulsifies  very  readily  and  evenly  so  that 
suspensions  for  vaccines  are  easily  made. 

It  is  a  well-known  fact  that  Pneumococcus  is  a  frequent  inhabitant  of  the  nasal, 
pharyngeal.  and  buccal  cavities.  The  explanation  of  infection  is  either  on  the 
ground  of  lowered  resistance  of  the  patient  or  enhanced  virulence  of  the  organism. 
Results  from  the  use  of  antipneumococcic  sera  have  been  of  only  slight  value.  Such 
sjira^are  useless  against  infections  with_other  strains.  Vaccines  appear  to  be  value-  >1- 
less  in  pneumonia  but  may  be  useful  in  local  infections.  They  probably  stimulate 
opsonin  production.  Oscar  Richardson  has  reported  an  organism  in  cases  of 
lobar  pneumonia,  cerebrospinal  meningitis,  mastoid  disease,  etc.,  bearing  resemb- 
lance to  both  pneumococci  and  streptococci — the  Streptococcus  capsulatus.  Other 
names  are  Streptococcus  mucosus  and  Pneumococcus  mucosus.  It  is  very  virulent 
for  mice  but  less  so  for  rabbits.  Park  isolated  it  twice  from  20  cases  of  pneu- 
monia. It  differs  from  the  Pneumococcus  in  that  the  colonies  on  blood-serum  are 
viscid  and  like  irregular  flecks  of  mucus.  The  characteristic  culture  is  a  glucose 
agar  stab.  (Reaction  must  not  exceed  +0.5.)  From  the  line  of  puncture  there  are 
flail-like  projections  extending  outward  from  ^  to  24  inch.  The  capsule  persists 
on  ordinary  culture  media.  This  organism  resembles  the  Streptococcus  of  Bonome 
of  the  French. 

In  a  study  of  blood  and  sputum  cultures  from  32  cases  of  lobar  pneu* 
monia  Hastings  and  Boehm  found  blood  and  sputum  positive  bacteriologically 
in  ii  cases.  In  nine  of  these  cases  the  Pneumococcus  was  isolated  and  in  2 
a  haemolysing  Streptococcus.  In  the  other  21  cases  the  sputum  cultures 
were  bacteriologically  positive  in  18  of  the  cases  and  negative  in  3.  In 
9  cases  the  Pneumococcus  was  isolated,  in  2  cases  B.  coli,  in  i  case  M.  catar- 
rhalis,  in  i  case  a  Staphylococcus,  in  2  cases  staphylococci  and  streptococci, 
in  i  case  B.  influenza.  The  percentage  of  positive  blood  cultures  was  30.3. 
Cole  obtained  30%  of  positive  blood  cultures.  The  blood  was  taken  into  flasks  of 
bouillon  in  dilution  of  1-50. 
5 


66 


STUDY   AND   IDENTIFICATION   OF  BACTERIA 


Diplococcus  Crassus. — This  is  a  Gram-positive,  kidney-shaped  dip- 
lococcus,  which  might  be  confused  with  the  M.  catarrhalis  or  the  Menin- 
gococcus  by  ordinary  staining  methods.  It  is  larger  than  the  Meningo- 
coccus. 

It  is  not  strongly  Gram-positive  as  one  may  find  examples  in  the  same  prepara- 
tion about  which  doubt  may  be  entertained.  It  ferments  lactose  and  saccharose 
as  well  as  glucose  and  maltose. 

In  throat  cultures  I  have  isolated  on  several  occasions  a  Gram-positive  diplococcus 
which  is  at  times  biscuit-shaped,  at  times  irregularly  spherical.  It  possesses  two 
or  three  metachromatic  granules,  so  that  in  a  Neisser  stain  for  diphtheria  the 
appearance  of  these  granules  may  be  confusing. 

Using  Ponder's  toluidin  blue  stain  I  have  observed  granule  staining  in  organisms 
of  round  or  oval  morphology  which  were  suggestive  of  the  ascospore  staining  of 
yeasts.  Staphylococci  may  show  granules  with  Ponder's  stain. 

Gram-negative  Cocci. — It  is  important  to  bear  in  mind  that  there 
are  many  cocci  of  varying  shapes,  which  in  cultures  or  in  smears  from 


FIG.  15. — Gonococcus.     Film  from  urethral  pus.     (Coplin.) 

the  throat,  nose  or  faeces  are  Gram-negative.  These  are  not  well  classi- 
fied or  described.  To  distinguish  the  three  important  kidney-shaped 
diplococci,  it  can  be  most  easily  accomplished  by  cultural  methods, 
using  hydrocele  agar  (ascites  or  blood  agar  will  answer),  ordinary  blood- 
serum  and  plain  agar.  The  Gonococcus  will  only  grow  on  the  hydro- 
cele agar;  the  Meningococcus  will  grow  on  this,  but  likewise  grows  on 


GONOCOCCUS  67 

ordinary  blood-serum.     The  M .  catarrhalis  will  grow  on  plain  agar  as 
well  as  on  other  media. 

Other  Gram-negative  organisms  of  confusing  morphology  are  M .  pharyngis  siccus, 
the  colonies  of  which  show  great  crinkly  dryness,  and  M.  pharyngis  flavus. 

Gonococcus  (Neisser,  1879). — This  organism  is  characteristically  a 
diplococcus,  the  separate  cocci  being  plano-convex  with  their  plane 
surfaces  apposed.  (Biscuit  shape,  coffee-bean  shape.)  They  are  gen- 
erally found  grouped  in  masses  of  several  pairs,  most  strikingly  in  pus 
cells  or  epithelial  cells,  but  also  found  extracellularly.  Except  in  the 
height  of  the  disease,  there  is  a  great  tendency  for  the  organisms  to 
show  involution  forms,  so  that  instead  of  biscuit-shaped  diplococci  we 
have  round,  irregular  and  uneven  cocci. 

It  is  therefore  advisable  in  searching  smears  from  chronic  gonorrhoea  to  continue 
the  search  of  Gram-stained  specimens  until  some  fairly  typical  diplococci  are  found. 
There  is  nothing  requiring  greater  discrimination  than  a  diagnosis  from  such  a  , 

smear.  At  the  commencement  of  a  gonorrhoea  the  epithelial  cells  are  abundant 
and  gonococci  are  found,  adhering  to  them  or  lying  free.  Later  on,  at  theacjmejpf  ^r 
the  discharge  (the  creamy,  abundant  discharge),  it  isjn  the  piis^cells  wj^md  them 
and  they  may  be  so  abundant  that  10  to  20%  of  the  pus  cells  may  contain  theni. 
In  the  subacute_stage  the  epjthelial  .cells,  which  practically  disappear  when  the 
discharge  is  so  abundant,  begin  tojreappear,  and  in  the  chronic  stage  the  epithelial 
cells_are  the  chief  ones,  and  are  the  ones  on  which  we  find  an  occasional  gonococcus, 
often  distorted  in  shape.  In  gonorrhceal  ophthalmia  the  gonococci  may  show 
appearances  in  the  conjunctival  epithelium  resembling  the  inclusion  bodies  of 
trachoma. 

The  best  method  of  diagnosis  in  cases  of  chronic  gonorrhoea  is  to  have  the  patient 
eat  the  stimulating  food  previously  interdicted,  to  take  active  exercise  and  to  have 
a  sound  passed.  To  obtain  material  for  examination  the  glans  penis  should  be 
washed  and  the  patient  who  has  presented  himself  with  a  full  bladder  should  pass 
a  portion  of  the  contained  urine.  Next  the  prostate  and  seminal  vesicles  should  be 
massaged  with  the  patient  standing  but  bent  over  and  the  penis  pendant.  The 
drops  of  discharge  from  the  massage  should  be  received  in  a  small  Petri  dish  and 
finally  the  remaining  urine  should  be  passed  into  a  sterile  bottle.  Smears  and 
cultures  should  be  made  from  the  sediment  of  the  two  urinary  specimens  and 
from  the  secretions  of  the  massaged  prostate  and  vesicles. 

The  smears  made  from  the  resulting  discharge  or  centrifuged  urine  will  probably 
contain  gonococci  if  they  are  present  in  the  urethra.  In  the  female  the  favorite 
sites  are  the  urethra  and  the  cervix  uteri.  In  municipal  examinations  it  is  customary 
to  make  two  smears:  one  from  the  urethral  meatus  and  a  second  from  the  cervix. 
The  vagina  is  not  a  suitable  soil  for  their  development.  In  female  children  it  is 
most  often  found  in  the  discharge  of  the  vulvovaginitis.  Gram-stained  smears  from 
pus  sediments  of  urine,  especially  in  pyelitis  or  cystitis,  may  show  coccoid  forms  of 
B.  coli,  which  may  be  phagocytized  and  thus  be  reported  as  gonococci. 


68  STUDY  AND   IDENTIFICATION  OF  BACTERIA 

In  addition  to  the  genital  organs,  the  Gonococcus  may  at  times  invade  and  be 
isolated  from  the  eye  (gonorrhceal  ophthalmia),  the  joints,  rarely  as  a  cause  of 
endocarditis  and  possibly  as  the  factor  in  septicaemia.  Grown  upon  hydrocele  or 
ascites  agar,  or  blood-streaked  agar,  or  upon  blood  agar  from  man  or  the  rabbit, 
the  colonies  appear  as  irregular,  minute,  dew-droryipots.  By  the  second  or  third 
day  the  involution  forms  are  abundant,  and  within  four  to  seven  days  the  culture 
will  probably  be  found  to  be  dead.  Unless  frequent  transfers  are  made,  it  will  be 
best  kept  alive  on  blood  agar.  The  organism  grows  best  at  37°C.,  and  will  not 
grow  below  25°C.  It  will  not  grow  on  plain  or  glycerine  agar  or  ordinary  blood- 
serum  unless  the  transfer  of  considerable  pus  in  inoculating  the  slants  gives  it  a 
suitable  culture  medium.  In  material  from  joints,  it  is  in  the  fibrin  flakes  that  the 
gonococci  are  most  apt  to  be  found,  if  found  at  all. 

Animals  do  not  contract  gonorrhoea.  Even  in  monkeys  urethral  in- 
oculations of  gonococci  are  negative.  The  organism  is  killed  in  five 
hours  by  a  temperature  of  45°C.  and  speedily  by  drying.  In  moist 
smears  of  pus  it  may  live  for  one  or  two  days. 

By  heating  the  blood-streaked  agar  tubes  to  s6°C.  for  twenty  minutes  (inactiva- 
tion-destroying  complement  and  hence  bactericidal  power  of  blood  on  slaflt)  greater 
success  in  primary  cultures  will  be  obtained. 

In  culturing  gonococci  the  transfer  of  material  to  culture  media 
should  be  made  with  the  least  delay  possible. 

The  most  satisfactory  medium  is  Thalman's  medium  upon  the  slanting  surface 
of  which  we  have  deposited  two  or  three  drops  of  human  serum.  Blood  may  be 
taken  from  a  vein  or  the  Wright  U  tube  may  be  used  and  after  centrifuging  the 
sterile  serum  is  taken  off  with  a  capillary  bulb  pipette  and  deposited  on  and  smeared 
out  on  the  slant.  We  now  use  Vedder's  starch  medium. 

Diplococcus  Intracellularis  Meningitidis  (Weichselbaum,  1887).— 
This  is  the  organism  of  epidemic  cerebrospinal  meningitis,  and  is  fre- 
quently termed  the  Meningococcus.  The  diplococcus  is  Gram-negative 
and  biscuit-shaped  and  is,  like  the  Gonococcus,  chiefly  contained  in  pus 
cells.  It  is  also  found  free  in  the  cerebrospinal  fluid  withdrawn  from 
cerebrospinal  fever  cases.  There  is  a  greater  tendency  to  variation 
in  size  and  shape  than  is  the  case  with  the  Gonococcus,  which  latter,  in 
fresh  material,  shows  a  striking  uniformity  morphologically.  The 
Meningococcus  is  at  times  not  abundant.  Early  in  the  case,  however, 
the  picture  may  be  similar  to  that  of  gonorrhceal  smears. 

On  blood-serum  the  colonies  appear  after  twenty-four  to  forty-eight  hours  as 
discrete,  very  slightly  hazy  colonies,  about  Ho  mch  in  diameter.  On  serum 
agar,  as  ascites  or  hydrocele  agar,  they  grow  best  and  show  as  faint  bluish 
colonies  about  i  to  2  mm.  in  diameter.  They  are  larger  than  Streptococcusjzi 
Pneumococcus  colonies.  Unless^  considerable  cerebrospinal  fluid  is  transferred 


THE  MENINGOCOCCUS  69 

with  the  inoculating  loop,  they  do  not  grow  on  plain  agar.  They  will  grow  at  times 
on  glycerine  agar.  The  organism  is  very  sensitive  to  light,  cold  and  drying.  It 
ferments  glucose  and  maltose  but  not  lactose  or  saccharose  and  only  grows  at  blood 
temperature,  thus  distinguishing  it  from  the  M.  catarrhalis  which  will  not  ferment 
any  of  these  sugars.  It  is  scarcely  pathogenic  for  laboratory  animals,  with  the 
exception  of  the  mouse  and  guinea-pig,  when  intraperitoneal  injections  but  not 
subcutaneous  ones  give  results.  Intradural  injections  give  results.  The  cultures 
die  out  very  rapidly,  so  that  it  is  necessary  to  make  transfers  every  one  or  two 
days.  The  Meningococcus  has  been  isolated  from  the  nasal  secretions  of  patients. 
The  possibility  of  these  organisms  being  the  M.  catarrhalis  must  be  considered. 
Of  the  greatest  importance  is  the  examination  of  the  naso-pharyngeal  material 
of  those  who  have  been  in  contact  with  a  patient.  These  healthy  carriers  are  im- 
portant. To  examine  such  people  introduce  a  bent  wire  applicator  with  sterile 
cotton  tip  past  the  soft  palate  so  as  to  get  material  from  the  naso-pharynx.  The 
material  should  be  immediately  inoculated  on  blood  or  serum  agar  and  quickly  put 
in  the  37°C.  incubator. 

Flexner  has  shown  that  in  monkeys,  which  are  susceptible  to  the  disease,  in-^ 
jections  of  cultures  of  M.  intracellularis  into  the  spinal  canal  is  followed  by  migra- 
tion of  the  cocci  to  the  nasal  cavity  both  free  and  in  phagocytic  leukocytes. 

The  Meningococcus  has  a  very  slight  resistance  to  sun  or  drying  so 
that  its  aerial  transmission  seems  doubtful.  It  is  supposed  to  effect  an 
entrance  by  the  nares,  thence  reaching  the  cerebral  meninges.  Infec- 
tion is  probably  by  direct  contagion.  Several  cases  have  been  reported 
where  with  a  high  leukocytosis  the  cocci  have  been  found  in  the  poly- 
morphonuclears  of  blood  smears  and  in  cultures  from  the  blood.  (In 
about  25%  of  blood  cultures  where  from  5  to  10  c.c.  are  employed.) 

By  the  use  of  initial  injections  into  horses  of  killed  cultures  followed  by  alternate 
injections  into  horses  of  living  diplococci,  then  seven  days  later  of  an  autolysate 
made  from  different  strains;  seven  days  later  again  injecting  living  diplococci;  thus 
alternating  material  every  week,  an  antiserum  of  value  has  been  obtained  by  Flexner. 
The  immunization  requires  about  one  year.  In  using,  withdraw  about  20  c.c.  of 
patient's  cerebrospinal  fluid  with  a  syringe,  and  then  inject,  through  the  same  needle, 
an  equal  quantity  of  the  serum.  The  injection  is  repeated  every  day  for  three  or 
four  days. 

As  the  result  of  extensive  study  of  the  relation  of  the  Meningococcus  to  cerebro- 
spinal fever  in  connection  with  epidemics  in  the  European  war  zone  many  authorities 
consider  the  causal  relation  of  the  organism  to  the  disease  as  unproven.  It  is  stated 
that  intraperitoneal  injection  of  huge  numbers  of  meningococci,  cultivated  in  the 
cerebrospinal  fluid,  failed  to  cause  symptoms  in  monkeys  while  intraperitoneal 
injection  of  a  filtrate  of  fluid  just  removed  from  a  patient  caused  symptoms  in  the 
monkey  similar  to  cerebrospinal  fever. 

The  history  of  hog  cholera  is  cited  by  Hort  and  others  to  show  that  our  long-stand- 
ing views  as  to  the~relstibnship  of  the  hog  cholera  organism  (B.  aertryk]  had  to  be 
abandoned  in  favor  of  a  filterable  virus  etiology,  The  possibility  of  a  filterable 
virus  being  the  cause  of  cerebrospinal  fever  is  suggested  but  the  point  is  emphasized 


70  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

that  the  presence  of  meningococci  in  a  person  has  a  value  as  indicating  the  presence 
of  the  virus,  so  that  such  examinations  should  continue,  but  they  insist  upon  the 
futility  of  means  for  eradicating  meningococci  from  the  nasopharynx  by  disin- 
fecting'solutions. 

For  diagnosis,  make  smears  and  cultures  from  cerebrospinal  fluid. 
The  sediment  from  the  centrifuged  material  gives  better  results.  In 
tuberculosis  the  lymphocytes  preponderate;  in  cerebrospinal  meningitis 
the  polymorphonuclears. 

It  has  been  stated  that  a  point  of  difference  between  the  phagocytosis  with  the 
gonococci  and  the  meningococci  is  that  the  meningococci  invade  and  at  times  destroy 
the  nucleus  of  the  polymorphonuclear,  which  is  not  true  of  gonococci.  The  appear- 
ance of  large  phagocytic  endothelial  cells,  often  containing  polymorphonuclears,  in 


FIG.  1 6. — Diplococctis  intracdlularis  meningitidis  and  pus  cells.     (Xiooo.) 

(Williams.} 

the  centrifuged  cerebrospinal  fluid  is  a  favorable  prognostic  sign.  At  times  there 
does  not  appear  to  be  any  relation  between  the  number  of  phagocytic  polymorpho- 
nuclears and  the  severity  of  the  case. 

Vincent  has  recommended  a  precipitin  test  for  epidemic  cerebrospinal  meningitis 
which  has  the  advantages  of  being  simple  and  more  immediate  than  cultures  and  of 
particular  value  in  those  cases  when  meningococci  cannot  be  found  in  the  smears  or 
in  cultures  from  the  cerebrospinal  fluid.  It  is  performed  by  adding  i  or  2  drops 
of  antimeningococcic  serum  to  a  tube  of  fresh  cerebrospinal  fluid  which  has  been 
clearecf  by  centrifugalization  for  ten  to  fifteen  minutes.  After  adding  the  serum  the 
tube  is  placed  in  the  incubator  at  S2°C.  for  two  to  five  hours  together  with  a  control 
tube.  The  formation  of  a  precipitate  (turbidity)  shows  a  positive  test. 

Micrococcus  Catarrhalis  (Seifert,  1890). — This  organism  has  been 
specially  studied  by  Lord.  It  resembles  the  Meningococcus  strikingly 
and  can  only  be  differentiated  by  cultural  procedures.  It  grows  on 


MALTA  FEVER  71 

plain  agar  and  at  room  temperature,  and  does  not  produce  acid  in  glu- 
cose media.  It  not  only  occurs  in  the  nasal  secretions  of  healthy  people, 
but  appears  to  be  responsible  for  certain  coryzas  and  bronchial  affec- 
tions, resembling  influenza.  It  also  is  responsible  for  certain  epidemics 
of  conjunctivitis. 

The  original  cultures  may  show  only  slight  growth  whereas  the  subcultures 
prove  luxuriant. 

The  colonies  are  larger,  more  opaque,  and  have  a  more  irregular  wavy  border 
than  the  round  colonies  of  the  Mening&coccus. 

The  colony  tends  to  be  easily  picked  up  from  the  plate  with  the  loop.     M.  catar-7~s; 
rhalis  grows  well  at  22° C.  after  several  days,  while  the  Meningococcus  requires  body^""" 
temperature.     It  does  not  ferment  with  acid  production  any  of  the  sugars. 

Micrococcus  Melitensis  (Bruce,  1887). — This  is  the  organism  of 
Malta  or  Mediterranean  fever,  sometimes  called  undulant  fever,  on 
account  of  successive  waves  of  pyrexia  running  over  several  months.  UJ 
The  disease  has  a  very  slight  mortality  (2%),  and  the  lesions  are  chiefly 
of  the  spleen,  which  is  large  and  diffluent.     The  organisms  can  best  be  (/j 
isolated  from  the  spleen  or  blood. 

M.  melitensis  is  only  about  0.3^1  in  diameter.  The  characteristics  are  its  very 
small  size  and  the  dew-drop  minute  colonies  on  agar,  which  at  incubator  temperature 
only  show  themselves  about  the  third  to  the  sixth  day.  It  is  nonmotile  and  Gram- 
negative.  In  bouillon  there  is  a  slight  turbidity.  Gelatine  growth  is  very  slow  and 
there  is  no  liquefaction.  Litmus  milk  becomes  more  blue  after  a  week  so  that  there 
is  an  alkaline  action.  Indol  is  not  produced.  The  optimum  reaction  of  media  is 
+  0.8.  It  grows  best  at  38°C. 

Many  laboratory  infections  have  been  recorded. 

The  organism  occurs  in  peripheral  circulation,  it  having  been  cultivated  from 
blood  very  successfully  by  Eyre.  He  takes  blood  at  the  height  of  the  fever,  and  in 
the  afternoon.  Formerly  it  was  customary  to  isolate  by  splenic  puncture. 

Infection  is  chiefly  by  means  of  the  milk  of  infected  goats.  The  organisms  are 
excreted  in  the  urine  of  patients,  and  a  diagnostic  point  is  to  make  plates  from  the 
urine.  Such  urine  applied  to  abraded  surfaces  causes  infection. 

The  serum  of  patients  shows  agglutinating  power  as  early  as  the  fifth  day  of 
the  disease,  and  this  may  persist  for  years  after  recovery.  Nicolle  has  advised  using 
serum  heated  to  56°C.  for  thirty  minutes  for  the  agglutination  test,  nonspecific  agglu- 
tinins  being  thereby  destroyed.  Carriers  may  be  of  importance  in  Malta  fever  and 
are  best  detected  by  agglutination  tests. 

A  high  mononuclear  increase  may  be  found  in  this  disease. 

Horses,  cows,  asses,  as  well  as  goats,  are  susceptible.  It  is  very  difficult  to 
infect  rabbits,  mice  and  guinea-pigs.  Monkeys  have  been  chiefly  utilized  in  ex- 
perimental work. 

It  would  appear  as  if  there  were  other  organisms  closely  related  to  M.  melitensis 
and  a  great  deal  is  now  being  written  as  to  confusing  serum  reaction  from  the  use 
of  M.  paramelitensist 


4 

72  STUDY  AND   IDENTIFICATION  OF  BACTERIA 

What  may  be  deemed  proof  positive  of  goats'  milk  transmission  is 
the  practical  disappearance  of  the  disease  among  the  naval  and  mili- 
tary forces  of  Malta,  as  the  result  of  boiling  the  milk,  while  still  con- 
tinuing among  native  civilians  not  boiling  their  milk.  Bassett-Smith 
has  noted  that  in  1905  there  were  798  cases  among  civilians  and  245 
naval  cases.  In  1907  there  were  457  cases  among  civilians  and  only 
twelve  cases  in  the  naval  forces. 

There  are  however  occasional  cases  which  Shaw  has  considered  as  due  to  carriers. 
As  the  organisms  are  excreted  in  faeces  as  well  as  in  urine,  and  as  the  course  of  the 
disease  is  so  protracted,  as  well  as  the  convalescence,  it  would  seem  that  the  carrier 
factor  should  be  of  more  importance  than  facts  would  justify. 

Mohler  has  noted  in  Texas,  where  the  disease  has  existed  for 
twenty-five  years,  that  the  Mexican  goatherds  boiled  their  milk  and 
hence  were  rarely  infected. 

The  souring  of  milk  does  not  destroy  the  germs  of  the  disease,  hence  transmission 
may  be  brought  about  by  butter  and  cheese. 

Malta  fever  was  stamped  out  of  Port  Said  by  destroying'all  infected  goats. 

Infection  may  occur,  i.  by  the  stomach  atrium  (usual),  2.  contam- 
inated dust  reaching  lungs,  3.  by  subcutaneous  infection. 


CHAPTER  VI 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.    SPORE- 
BEARING  BACILLI.    KEY  AND  NOTES 

A.  Grow  aerobically. 

1.  Stab  culture  in  gelatin  has  branches  growing  out  at  right  angles  to  line  of  stab, 
(a)  Has  no  membrane  on  bouillon  or  liquefied  gelatin.     Projecting  branches 

from  line  of  stab  only  at  upper  part  of  line  of  growth.    Absolutely  non- 
motile.     Ends  shaiplyjnit  across  or  concave.     ANTHRAX  GROUP. 
(6)  Has  thick  whitisfTniembrane  on  bouillon  and  surface  of  liquefied  gelatin. 
Projecting  branches  all  along  the  line  of  stab.     Sluggishly  motile.     MY- 
COIDES  GROUP.     (B.  mycoides.     B.  ramosus.)"" 

2.  Stab  cultures  in  gelatin  do  not  show  projecting  branches. 

(a)  Potato  cultures  do  not  become  wrinkled.  At  first  slightly  moist,  later 
dry  and  mealy.  SUBTILIS  GROUP.  (Hay  bacillus  group.)  Actively 
motile  with  more  or  less  square  ends  and  a  central  spore  which  is  of  the 
same  diameter  or  only  slightly  larger  than  the  bacillus.  The  yellow  sub- 
tilis  is  at  times  found  in  water.  The  colonies  on  potato  are  of  a  cheese- 
yellow  color.  The  bacilli  are  very  large  and  show  a  sluggish,  worm-like 
motion. 

B.  megatherium  often  shows  a  granular  or  beaded  appearance  in  a  Gram 
preparation.  The  narrow  spores  are  never  central,  usually  between  center 
and  end,  and  rather  elongated.  It  most  nearly  resembles  the  sporulating 
bacillus  of  malignant  oedema  but  if  the  spore  is  quite  terminal  and  bulging 
may  resemble  B.  tetani. 
Cultures  of  B.  megatherium  are  somewhat  similar  to  B.  coli  colonies. 

(&)  Potato  cultures  at  first  even  growth  but  after  a  few  days  become  wrinkled. 
VULGATUS  GROUP.  (Potato  bacillus.) 

B.  vulgatus  shows  marked  wrinkling,  like  intestinal  coils.     B.  mesentericus 
show  slight  wrinkling  and  a  network-like  appearance. 
Two  water  bacilli  belonging  to  this  group  are  the  B.  mesentericus  fuscus 
(brown  growth)  and  B.  mesentericus  ruber  (red  growth). 

NOTE. — The  following  cultural  characteristics  are  common  to  all  the  above  spore 
bearers. 

1.  Liquefaction  of  gelatin. 

2.  Milk  slowly  and  incompletely  coagulated  with  very  little  change  in  reaction. 
Later  the  coagulum  is  digested. 

3.  No  gas  in  either  glucose  or  lactose. 

4.  No  indol. 

5.  All  are  Gram-positive. 

6.  All  digest  blood-serum.   ^^ 

W  73 


74 


STUDY   AND   IDENTIFICATION   OF  BACTERIA 


NONPATHOGENIC    SPORE-BEARING    AEROBES    ON   AGAR 
Modification  of  table  of  Gruner  and  Fraser. 


Gray  white 


1  Chalk  like 

(  Edges  feathery 

Surface  gummy     f  Yellow 

\  White 

Yellow 

Surface  moist 

Dirty  gray 

and  exuberant 

White  and 

wrinkled. 

B.  subtilis  (motile). 

B.  ellenbachensis  (non-motile). 

B.  cretaceus. 

B.  mycoides. 

B.  ochraceus. 

B.  mesentericus. 

B.  ruminatus. 

B.  graveolens,. 

B.  vulgatus. 


NOTES. — B.  subtilis  has  square  ends  and  central  spores,  not  causing  bulging. 

B.  megatherium  has  spore  toward  one  pole  which  may  be  terminal. 

B.  vulgatus  is  long  and  slender.     Slightly  oval  spores. 

B.  mesentericus  varies.  Usually  short  with  rounded  ends  and  central  bulging 
spores. 

B.  ellenbachensis  has  rounded  ends,  oval  spores  and  shows  granule  formation 
(beaded) — resembling  diphtheroids. 

All  of  these  grow  well  at  room  temperature,  optimum  3o°C. 
B.  Grow  only  anaerobically. 

1.  Rods  very  little  swollen  by  centrally  situated  spores, 
(a)  Motile.     B.  cedematis  maligni.     (Gram-negative.) 
(6)  Nonmotile.     B.  aerogenes  capsulatus.     (Capsule.) 

2.  Spores  tend  to  be  situated  between  center  and  end. 
(a)  No  liquefaction  of  gelatin.     B.  butyricus. 

(6)  Gelatin  liquefied  slowly. 

B.  botulinus.     Milk  not  coagulated. 

B.  anthracis  symptomatici. 

B.  enteritidis  sporogenes.     Milk  coagulated  with  abundant  gas. 
(c)  Gelatin  liquefied  rapidly.     B.  cadaveris  sporogenes.     Very  motile. 

3.  Spores  situated  at  end  of  rod.    Drum-stick  sporulation.    TETANUS  GROUP. 

The  following  table  taken  from  Lehmann  and  Neumann,  based  on 
pathogenic  effects,  is  of  great  practical  value.  After  inoculation  of 
some  animal  subcutaneously  with  the  suspected  material  we  have: 

A.  No  particular  symptoms  at  site  of  inoculation. 

Absorption  of  the  soluble  toxin  causing: 

(1)  General  symptoms  of  tetanus.     B.  tetani. 

(2)  Botulism    poisoning    symptoms.     Pupillary    symptoms.     Paralysis     of 
tongue  and  pharynx.     Cardiac  and  respiratory  failure. 

B.  Local  symptoms  marked  at  site  of  inoculation.     Hemorrhagic  emphysema tous 
oedema. 

(i)       Motile. 

(a)  Gram-negative.  B.  cedematis  maligni. 

(6)  Gram-positive.  ^anthracis  symptomatici. 


ANTHRAX 


75 


(2)       Nonmotile. 

B.  aerogenes  capsulatus,  B.   phlegmonis   emphysematosse  (Frankel)   or 
B.  perfringens. 

SPORE-BEARING  AEROBES 

Bacillus  anthracis  (Pollender  discovered  1849.  Davaine  recognized 
nature  1863.  Koch  proved  1876).— Of  the  aerobic_spore-bearing 
bacilli  this  is  the  only  one  of  particular  medical  importance"" 

Anthrax  is  an  important  disease  in  domesticated  animals,  especially  sheep  andcattle. 
The  characteristic  postmortem  change  in  animals  is  the  greatly  enlarged,  friable, 
mushy  gpleen.  Man  is  much  less  susceptible  than  these  animals,  but  is  more  so 
than  the  goat,  horse,  or  pig.  The  41gerian^sheep  has  a  high  degree  of  immunity, 


FIG.  17. — Anthrax  bacilli.     Cover-glass  has  been  pressed  on  a  colony  and  then  fixed 
and  stained.     (Kolle  and  Wassermann.) 

as  has  the  white  rat.  The  brown  rat  is  quite  susceptible  as  are  also  guinea-pigs,  mice 
and  rabbits.  The  disease  in  man  chiefly  occurs  among  those  working  with  hides, 
wool,  or  meat  of  infected  cattle.  The  two  chief  types  in  man  are:  i.  Malignant 
pustule  and  2.  Woolsorter's  disease.  An  intestinal  type  is  also  recognized. 

Malignant  pustule  results  from  the  inoculation  of  an  abrasion  or  cut; 
thus  it  frequently  shows  on  the  arms  and  the  backs  of  those  unloading 
hides.  It  first  appears  as  a  pimple,  the  center  of  which  becomes  vesicu- 
lar, then  necrotic. 

A  ring  of  vesicles  surrounds  this  central  eschar  and  a  zone  of  congestion,  the 
vesicles.  The  lymphatics  soon  become  inflamed  as  well  as  neighboring  glands. 
If  the  pustule  is  not  excised  andtleath  occurs,  there  is  not  much  enlargement  of  the 


STUDY  AND   IDENTIFICATION  OF  BACTERIA 


spleen  and  the  bacteria  are  not  abundant  in  the  kidneys,  etc.,  as  with  animals. 
Man  seems,  to  die  from  toxaemia  rather  than  a  septicaemia. 

In  woolsorter's  disease  there  is  great  swelling  and  oedema  of  the  bronchial 
and  mediastinal  glands.  The  lungs  show  oedema,  which  about  the  bronchi  is 
hemorrhagic. 

The  bacillus  is  5  to  8p  by  i  to  ij^/i  and  nonmotile.  In  cultures  it 
has  square  cut  or  concave  ends  and  is  often  found  in  chains,  but  in  the 
blood  of  an  infected  animal  the  free  ends  of  the  rods  are  somewhat 
rounded.  It  is  Gram-positive.  Colonies,  by  interlacing  waves  of 
strings  of  bacteria,  show  Medusa  head  appearance.  For  cultural  char- 
acteristics see  key.  Spores  develop  best  at  a  temperature  of  3o°C.  and 


FIG.  1 8. — Anthrax  bacilli  growing  in  a  chain  and  exhibiting  spores.     (Kolle  and 

Wassermann.) 


do  not  form  at  temperatures  above  43°C. 
placed.     They  stain  with  difficulty. 


They  are  oval  and  centrally 


• 


Stiles  thinks  that  animals  are  infected  by  eating  the  bones  of  animals  which  have 
died  of  anthrax,  cutting  buccal  mucous  membrane,  and  so  becoming  infected. 
Spores  do  not  form  in  an  intact  animal  body,  but  they  do  form ^  after ji  postmortem 
or  the  disintegration  of  the  body  by  maggots.  For  this  reason  it  is  better  not  to 
open  up  the  body  of  the  animal,  but  to  make  the  diagnosis  by  cutting  off  an  ear. 
Dried  spores  will  live  for  years  and  will  withstand  boiling  temperature  for  hours. 

In  vaccinating  animals  against  anthrax,  Pasteur  used  two  vaccines.  The  first 
is  attenuated  fifteen  days  at  42.5°C.  The  second,  attenuated  for  only  ten  days,  is 
given  twelve  days  later. 

Various  bacteria,  especially  B.  pyocyaneus,  show  marked  antagonism  to  B. 
^anthracis.  Pyocyanase  digests  the  anthrax  bacillus  and  has  been  used  to  cure 

iN        ot-iirviolc    info^i-orl    TirifV*    *»  TifV»  TO  v 


animals  infected  with  anthrax. 


SYMPTOMATIC  ANTHRAX 


77 


In  taking  material  from  a  malignant  pustule  before  excision,  be  care* 
ful  not  to  manipulate  it  roughly,  as  bacteria  may  enter  the  circulation. 
Make  cover-glass  preparations,  staining  by  Gram.  Make  culture  on 
agar.  Blood  cultures  are  usually  only  positive  later  in  the  disease. 
Inoculate  a  guinea-pig  or  a  mouse  subcutaneously. 

The  guinea-pig  dies  in  about  forty-eight  hours  and  shows  an  oedematous  gelatinous 
exudate  at  site  of  inoculation.  The  blood  is  black  and  swarms  with  anthrax  bacilli. 
It  is  the  best  example  of  a  septicaemia. 

Anorganism  with  a  central  spore  and  morphologically  resembling  B.  anthracis, 
but  motile,  has  been  reported  as  occurring  in  the  stools  of  pellagrins.  Gelatin  stabs 
show  a  cup-shaped  liquefaction  in  about  five  days.  No  change  in  milk.  The 


FIG.  19. — Bacillus  anthracis  in  blood  of  rabbit.     (Coplin.) 

colonies  are  slimy  and  opaque.     The  organism  is  said  to  be  agglutinated  by  the 
serum  of  pellagra  cases.     The  name  B.  maydis  has  been  given  to  it. 

There  is  an  anaerobic  spore  bearer,  called  the  bacillus  of  symptomatic 
anthrax,  blackleg  or  quarter-evil,  which  causes  a  rapidly  developing 
emphysematous  swelling,  with  a  dark  color  of  the  thighs.  It  is  called 
B.  chauwei.  It  has  bulging,  slightly  oval  spores  at  one  end,  but  they 
are  not  distinctly  terminal  as  with  tetanus  spores.  It  affects  sheep 
and  cattle  but  not  man.  It  is  a  soil  organism  like  those  of  tetanus, 
malignant  oedema  and  gas  gangrene. 

There  is  also  an  aerobic  spore-bearing  bacillus  called  B.  anthracoides, 
which  differs  morphologically  from  anthrax  solely  in  its  rpunded_erids 
jn Culture.  Its  growth  is  more  rapid  and  it  liquefies  gelatin'  more 
energetically. 


78  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

SPORE-BEARING  ANAEROBES 

There  are  four  very  important  pathogens  in  this  group — that  of  gas 
gangrene;  that  of  malignant  oedema;  that  of  botulism,  and  the  organism 
of  tetanus. 

The  B.  enteritidis  sporogenes  is  of  importance  in  connection  with  indications  of 
faecal  contamination  of  water.  In  connection  with  B.  aero  genes  capsulatus,  there  is 
some  question  as  to  whether  the  extensive  oedema  produced  by  it  may  not  usually 

be  from  a  terminal  or  cadaveric  infec- 
tion. At  any  rate  necrotic  material 
seems  necessary. 

It  should  be  stated  that  our  knowl- 
edge of  the  differential  cultural  charac- 
teristics of  anaerobes  is  unsatisfactory. 
The  exact  methods  which  are  in  use 
for  aerobes  have  not  been  applied  to 
anaerobic  organisms. 

To  Cultivate  Anaerobes. — Prob- 
ably the  apparatus  giving  the  most 
perfect  anaerobic  conditions  is  the 
Novy  jar,  in  which  the  air  has  been 
replaced  by  hydrogen.  The  diffi- 
culties attending  the  method  are: 

i.  Unless  a  special  apparatus  (Kipp's) 
is  at  hand,  there  may  be  difficulty  in  pre- 
FIG.  20. — Novy  jar.  venting  the  sulphuric  acid  from  frothing 

over  when  poured  on  the  zinc.    It  should, 
at  first,  be  added  in  small  quantities  at  a  time — well  diluted  (i  to  6). 

2.  Various  wash-bottles  are  required:  one  containing  silver  nitrate  solution  for 
traces  of  AsH3  and  one  with  lead  acetate  for  H2S  and  another  with  pyrogallic  acid 
and  caustic  soda  for  any  oxygen  that  may  come  over. 

3.  Mixtures  of  hydrogen  and  air  explode.     Consequently,  in  determining  whether 
all  air  has  been  expelled  and  in  its  place  an  atmosphere  of  hydrogen  exists,  it  is 
necessary  to  see  if  the  escaping  gas  burns  with  a  blue  flame.     Unless  this  is  collected 
in  a  test-tube  and  examined,  we  may  have  an  explosion. 

4.  Except  in  a  large  laboratory,  where  the  apparatus  is  set  up  and  ready  for  use, 
too  much  time  would  be  required. 

5.  Simpler  methods  appear  to  give  as  good  results. 

In  Tarozzi's  method,  pieces  of  fresh  sterile  organs  are  added  to 
bouillon.  Pieces  of  kidney,  liver,  or  spleen  are  best  suited.  After 
adding  the  tissue  the  media  may  be  heated  to  8o°C.  for  a  few  minutes 


CULTTJRING  ANAEROBES 


79 


without  interfering  with  the  anaerobic  condition  producing  properties 
of  the  fresh  tissues.  This  method  is  practically  the  same  as  that  recom- 
mended by  Smith  (see  Tetanus).  This  is  also  a  feature  of  Noguchi's 
method  of  culturing  Treponema  pallidum. 


The  Method  of  Liborius 

In  this  it  is  necessary  to  have  a  test-tube  containing  about  4  inches  of  a  i% 
glucose  agar.  Glucose  acts  as  a  reducing  agent  and  furnishes  energy.  It  is  con- 
venient to  add  about  i/io  of  i%  of  sulphindigotate  of  soda;  the  loss  of  the  blue  color 
at  the  site  of  the  colony  enabling  us  to  pick  them 
out.  The  tube  of  agar  should  be  boiled  just  be- 
fore using'  to  expel  remaining  oxygen  from  the 
tube.  Now  rapidly  bring  down  the  temperature 
to  about  42°C.,  by  placing  the  tube  in  cold  water, 
and  inoculate  the  material  to  be  examined.  A 
second  or  third  tube  may  be  inoculated  from  the 
first,  just  as  in  ordinary  diluting  methods  for  plate 
cultures.  Having  inoculated  the  tubes,  solidify 
them  as  quickly  as  possible,  using  tap  water  or 
ice-water.  The  anaerobic  growth  develops  in  the 
depths  of  the  medium.  Some  pour  a  little  sterile 
vaseline  or  paraffin  or  additional  agar  on  the  top 
of  the  medium  in  the  tube  as  a  seal  from  the  air. 
Others  have  recommended  the  inoculation  of  some 
aerobe,  as  B.  prodigiosus,  on  the  surface.  This 
latter  method  is  not  advisable.  A  deep  stab 
culture  is  often  sufficient. 

The  same  technic  can  be  applied  to  gelatin 
cultures  for  anaerobes,  pouring  in  at  the  comple- 
tion of  the  inoculation  an  inch  or  so  of  melted 
glucose  agar  to  act  as  a  stopper  for  the  gelatin 
layer  below. 


FIG.  21. — Arrangement  of 
tubes  for  cultivation  of  anae- 
robes by  Buchner's  method. 
(Mac  NeaL) 


The  Method  of  Buchner 

In  this  method  i  gram  each  of  pyrogallic  acid 
and  caustic  potash  or  soda  for  every  100  c.c.  of 
space  in  the  vessel  containing  the  culture  is  used 
to  absorb  the  oxygen.  It  is  convenient  to  drop 
in  the  pyrogallic  acid;  then  put  in  place  the  in- 
oculated tubes  or  plates;  then  quickly  pouring  in  the  amount  of  caustic  soda,  in  a 
10%  aqueous  solution,  to  immediately  close  the  containing  vessel.  A  large  test- 
tube  in  which  a  smaller  one  containing  the  inoculated  medium  is  placed,  and  which 
may  be  closed  by  a  rubber  stopper,  is  very  convenient.  A  good  rubber-band  fruit 
jar  is  satisfactory.  A  desiccator  may  be  used  for  plates.  An  excellent  method  for 


80  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

anaerobic  plates,  either  in  a  desiccator  with  the  pyrogallic  acid  and  caustic  soda,  or 
less  satisfactorily  in  the  open  air,  is  to  sterilize  the  parts  of  the  Petri  dish  inverted; 
that  is,  the  smaller  part  is  put  bottom  downward  in  the  inverted  cover  (as  one  would 
set  one  tumbler  in  another).  Then,  in  using,  unwrap  the  Petri  dish,  lift  up  the  inner 
part,  pour  in  the  inoculated  medium  into  the  upturned  cover.  Then  immediately 
press  down  the  inner  dish,  spreading  out  a  thin  film  of  the  medium  between  the  two 
bottoms. 

Zinsser  has  originated  a  very  satisfactory  method  for  plate  cultures  of  anaerobes, 
which  is  shown  in  Fig.  8. 

Secure  two  small  crystalh'zing  dishes,  about  3  and  4  inches  in  diameter  by  i  inch 
depth  and  sterilize  as  for  Petri  dishes.  Pour  the  inoculated  agar  into  the  smaller 
of  the  dishes  or  one  can  smear  the  surface  of  poured  glucose  agar  with  the  material 
to  be  plated  out.  In  the  bottom  of  the  larger  dish  place  the  dry  pyrogallic  acid, 
then  invert  the  smaller  dish  with  the  agar  surface  over  it.  Quickly  pour  a  5% 
solution  of  caustic  soda  into  the  space  separating  the  sides  of  the  inverted  smaller 
dish  and  the  upright  larger  dish,  to  a  depth  of  %  inch  and,  as  it  is  dissolving  the 
pyrogallic  acid,  very  speedily  superimpose  paraffin  oil  on  the  soda  solution  to  make 
an  air-tight  seal. 

J.  H.  Wright's  Method 

Make  a  deep  stab  culture  in  glucose  agar  or  gelatin,  preferably  boiling  the  media 
before  inoculating.  Then  flame  the  cotton  plug  and  press  it  down  into  the  tube  so 
that  the  top  lies  about  three-fourths  of  an  inch  below  the  mouth  of  the  test-tube. 
Next  fill  in  about  one-fourth  of  an  inch  with  pyrogallic  acid;  then  add  2  or  3  c.c.  of 
a  10%  solution  of  caustic  soda,  and  quickly. insert  a  rubber  stopper.  This  method 
is  one  of  the  most  convenient  and  practical,  and  is  to  be  strongly  recommended. 

Method  of  Vignal 

In  this  a  section  of  glass  tubing  (1/4  in.)  is  drawn  out  at  either  end,  as  in  making  a 
bacteriological  pipette,  with  a  mouth-piece  containing  a  cotton  plug.  The  liquid 
agar  or  gelatin  is  then  inoculated  and  the  medium  drawn  up  into  the  tube  by  suction 
with  mouth  or  better  with  a  rubber  bulb.  In  a  very  small  flame  the  capillary  nar- 
rowings  are  sealed  off,  and  we  have  inside  the  tube  very  satisfactory  anaerobic  con- 
ditions. To  get  at  the  colonies,  file  a  place  on  the  tube  and  break  at  this  point. 

To  obtain  material  for  examination  and  isolation  in  pure  culture  from  the  deep 
agar  stab-tube,  it  is  best  to  loosen  the  medium  at  the  sides  of  the  tube  with  a  heated 
platinum  spud  or  a  flattened  copper  wire.  Then  shake  the  mass  out  into  a  sterile 
Petri  dish.  It  is  dangerous  to  break  the  tubes  with  a  hammer  as  some  do. 

With  those  anaerobes  which  produce  gas  in  glucose  agar  the  split  in  the  column 
of  medium  enables  one  to  introduce  a  fine  sterile  capillary  pipette  to  the  site  of  a 
colony  and  by  releasing  pressure  on  the  rubber  bulb  to  draw  up  into  the  tip  of  the 
tube  material  for  investigation. 

A  Combination  Method 

Recently  as  shown  in  the  illustration  in  Fig.  7,  I  have  been  combining  various 
methods  so  that  very  satisfactory  anaerobic  conditions  are  obtained.  First,  a 


GAS  GANGRENE  8 1 

deep  agar  stab  of  freshly  sterilized  glucose  agar  is  made  and  inoculated.  The  sur- 
face of  this  is  then  covered  with  sterile  paraffin  oil.  The  proper  amount  of  pyro- 
gallic  acid  is  then  deposited  in  a  salt  mouth  bottle.  The  rubber  stopper  with  the 
glass  and  rubber  tubing  is  then  firmly  pushed  in  and  connection  made  with  a  filter 
pump. 

In  five  to  ten  minutes  almost  all  the  air  will  be  exhausted  when  the  Hofmann 
clamp  is  screwed  up  tight  and  the  bottle  disconnected  from  the  vacuum  pump. 
The  glass  tubing  end  is  then  inserted  into  a  graduate  holding  10%  caustic  soda 
solution,  the  Hofmann  clamp  unscrewed,  and  the  necessary  amount  of  caustic  soda 
having  been  run  in,  as  noted  under  Buchner  method,  we  again  close  the  screw  clamp 
and  incubate. 

GENERAL  CONSIDERATIONS  OF  PATHOGENIC  ANAEROBES 

Dean  and  others,  working  with  gas  gangrene  wounds,  have  brought 
out  some  very  practical  points  in  connection  with  the  three  important 
anaerobes  found  in  such  wounds,  viz.:  B.  aero  genes  capsulalus,  B.  adem- 
atis  maligni  and  B.  tetani. 

They  found  an  egg  broth,  made  by  shaking  up  the  white  and  yolk  of  i  egg  in 
300  c.c.  water  a  most  excellent  culture  medium.  This  medium  was  tubed  and 
sterilized,  after  which  it  was  liberally  inoculated  with  material  from  the  wounds. 
After  inoculation  the  tube  was  heated  to  8o°C.  for  one-half  hour  and  then  incubated 
anaerobically..  The  gas  bacillus  was  present  in  abundance  in  such  cultures  after  two 
or  three  days'  incubation,  while  the  bacillus  of  malignant  oedema  later  on  became  the 
predominant  organism.  The  tetanus  bacillus  only  appeared  after  a  prolonged  period 
— seven  to  ten  days.  The  bacillus  of  malignant  oedema  grew  best  on  Dorsett's  egg 
medium  and  in  two  days  began  to  liquefy  the  slant  with  a  bluish-black  discolora- 
tion. The  growth  at  first  was  profuse  and  creamy  white.  On  glucose  agar  there 
was  much  less  gas  production  than  with  the  gas  bacillus.  The  malignant  oedema 
organisms  were  as  a  rule  Gram-negative  on  glucose  agar  but  they  were  distinctly 
Gram-positive  on  Dorsett's  medium.  The  spores  were  oval  in  shape,  usually 
located  near  the  end  of  the  rod.  The  gas  bacillus  grew  well  on  Dorsett's  medium 
but  less  vigorously.  In  glucose  agar  stabs  so  much  gas  was  formed  that  the 
cotton  plug  and  much  of  the  culture  medium  tended  to  be  expelled  from  the 
tube.  The  spores  form  well  on  Dorsett's  medium  but  not  in  glucose  agar  and 
show  as  oval  bodies  distending  the  central  portion  of  the  rod. 

Subcutaneous  inoculation  of  the  gas  bacillus  and  that  of  malignant 
oedema  rarely  produced  death  in  guinea-pigs. 

As  regarded  the  tetanus  bacillus  they  tried  various  methods  of  bacteriological 
diagnosis.  The  examination  of  smears  from  wounds  was  unsatisfactory  in  search 
for  "drum-stick"  spores.  In  broth  cultures  the  spores  were  not  present  until  after 
several  days  and  in  mixed  cultures  it  was  difficult  to  be  sure  that  the  terminal  spores 
were  those  of  tetanus  and  not  atypical  malignant  oedema  spores.  The  best  method 
6 


82  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

was  to  inject  guinea-pigs  in  the  subcutaneous  tissues  of  the  left  chest  with  i  or  2  c.c. 
of  mixed  broth  culture.  In  two  or  three  days  stiffness  of  the  left  forelimb  was  observed 
soon  becoming  quite  stiff  and  extended.  The  spasm  extends  to  other  limbs  and 
death  occurs  in  one  or  two  days.  There  is  no  evidence  of  marked  inflammation  at 
the  site  of  inoculation. 

There  have  been  a  number  of  cases  of  delayed  tetanus  often  asso- 
ciated with  operations  done  a  month  or  two  after  the  original  wound 
infection  so  that  it  is  recommended  to  give  antitetanic  serum  before 
operation  on  such  cases. 

B.  (Edematis  Maligni  (Pasteur,  1877). — This  is  the  vibrion  septique 
of  Pasteur.  It  is  found  in  garden  soil  and  in  street  sweepings.  It  is 
the  cause  of  an  acute  cellular  necrosis  attended  with  serous  sanguino- 
lent  exudation  and  with  more  or  less  emphysema.  The  organism  only 
becomes  generalized  in  the  blood  about  the  time  of  death  and  post- 
mortem. Therefore,  it  is  not  a  septicaemia,  as  is  anthrax.  The  bacil- 
lus is  an  organism  about  the  size  of  anthrax  (*jn  by  0.8),  but  is  narrower 
and  does  not  have  the  same  square  cut  or  dimpled  ends.  Furthermore, 
it  is  motile,  Gram-negative  and  an  anaerobe.  The  guinea-pig  is  very 
susceptible,  and  about  the  time  of  death  and  postmortem  there  may  be 
seen  long  flexile  motile  filaments,  15  to  40^1  long,  which  move  among 
the  blood-cells  as  a  serpent  in  the  grass  (Pasteur). 

In  cultures  it  grows  out  very  slightly  from  the  line  of  stab,  giving  a  jagged  granular 
line,  differing  from  tetanus.  Spores  form  best  at  37°C. — requiring  about  forty- 
eight  hours.  It  liquefies  gelatin.  In  examining  an  exudate  from  a  suspected  case- 
note  the  presence  of  spores  centrally  situated.  Inoculate  a  guinea-pig.  Death 
occurs  in  about  two  days.  There  is  intense  hemorrhagic  emphysematous  oedema  at 
the  site  of  inoculation,  the  cedematous  fluid  however  does  not  show  spores.  The 
bacilli  do  not  appear  in  the  blood  until  about  the  time  of  death  and  it  is  an  assistance 
in  diagnosis  to  put  the  dead  body  of  the  guinea-pig  in  the  incubator  for  a  few  hours. 
The  subcutaneous  tissue  contains  fluid  and  gas.  There  is  present  the  foul  odor  of 
an  anaerobe.  Examine  for  the  long  filaments  showing  flowing  motility.  Be  sure 
to  stain  by  Gram.  (Negative.)  For  cultures,  heat  the  material  (either  from  a 
wound  or  from  a  guinea-pig)  which  shows  spores  to  a  temperature  of  80° C.  for 
from  fifteen  minutes  to  one  hour.  Then  inoculate  glucose  agar  stab  culture  and  grow 
anaerobically.  Courmont  differentiates  anthrax  from  malignant  oedema  by  in- 
jecting into  ear-vein  of  rabbit.  The  injection  of  malignant  oedema  in  this  way, 
instead  of  subcutaneously,  tends  to  immunize. 

B.  Botulinus  (Van  Ermengem,  1896). — This  is  the  organism  which 
produces  botulism,  a  form  of  meat  poisoning.  It  is  a  spore-bearing 
anaerobe  and  must  not  be  confused  with  another  organism,  a  non- 
sporing  aerobic  bacillus,  associated  with  meat  poisoning — the  B. 


BOTULISM 


enteritidis  of  Gartner.     The  spores  are  at  the  end  and  are  not  very 
resistant;  a  temperature  of  8o°C.  often  killing  them. 


FIG.  22. — Bacillus  of  botulism.     (Kolle  and  Wassermann.) 

In  botulism  the  meat  becomes  infected  after  the  animal  has  been  slaughtered; 
in  Gartner  meat  poisoning  the  cow  meat  was  infected  at  the  time  of  slaughter — it 
was  from  a  sick  animal.  Thorough  cooking  of  the  meat  protects  against  botulism 
but  not  certainly  against  Gartner  meat  poisoning. 


FIG.  23. — Symptomatic   anthrax    (Rauschbrand)    bacilli   showing   spores.     (Kolle 

and  W  assermann.) 

There  are  dysphagia,  paralysis  of  eye-muscles,  and  cardiac  and  respiratory 
symptoms  (medulla).  The  symptoms  are  due  to  the  elaboration  of  a  soluble  toxin 
of  the  same  nature  as  that  of  diphtheria  and  tetanus.  There  is  no  fever  and  con- 
sciousness is  preserved. 


84  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

An  antitoxin  which  it  is  stated  has  therapeutic  value  in  botulism  has  been  pre- 
pared in  the  usual  way  by  Kempner.  Without  serum  treatment  death  occurs  in 
about  40%  of  cases  and  takes  place  between  twenty-four  and  forty-eight  hours. 

The  bacillus  has  been  isolated  from  sausage  and  ham.  It  is  a  rare  cause  of  food 
poisoning,  most  of  such  cases  being  the  result  of  paratyphoid  or  enteritidis  infections. 
It  is  a  large  bacillus — 5  to  ic/xXiM-  It  is  slightly  motile  and  stains  by  Gram. 
It  produces  gas  in  glucose  media.  It  grows  best  at  22°  and  only  slightly  at  37° — 
hence  it  is  dangerous  only  from  its  soluble  toxin,  the  bacilli  not  developing  to  any 
extent  in  the  body. 

For  this  reason  botulism  patients  are  not  a  source  of  danger,  it  is  the  infected 
meat  alone  which  causes  the  disease.  On  the  contrary  where  the  meat  poisoning 
is  due  to  the  Gartner  or  paratyphoid  group  infection  may  take  place  from  the 
patient's  discharges.  Botulism  is  an  intoxication,  not  an  infection. 

When  the  toxin  is  introduced,  it  requires  a  period  of  incubation  of 
twelve  to  twenty  hours.  Symptoms  of  gastrointestinal  disorder  may 
come  on  shortly  after  the  ingestion  of  the  toxin  containing  food,  these 
however  are  not  the  specific  manifestations,  as  are  the  eye  symptoms, 
etc. 

An  important  point  is  that  ham  may  not  appear  decomposed  and  yet  contain 
many  bacilli  and  much  toxin.  It  is  a  very  potent  toxin — as  little  as  Ho 00 
c.c.  may  kill  a  guinea-pig.  In  man  the  toxin  is  apparently  absorbed  from  the 
alimentary  canal,  thus  differing  from  most  toxins  as  well  as  venoms  which  are 
usually  harmless  when  introduced  by  mouth. 

For  diagnosis  inject  an  infusion  of  the  ham  or  sausage  which  was  eaten  of  into  a 
guinea-pig,  and  characteristic  pupillary  symptoms  with  death  by  cardiac  and 
respiratory  failure  will  result. 

Cultures  may  be  made  in  glucose  agar. 

The  culture  is  disrupted  by  gas.  Incubation  at  room  temperature  and  in  the 
dark  is  necessary.  There  is  a  rancid  odor.  The  characteristic  point  is  the  pro- 
duction of  a  powerful  soluble  toxin  which  produces  symptoms  when  no  bacilli  are 
present. 

B.  Tetani  (Nicolaier,  1885;  Kitasato,  1889).— This  is  the  most  im- 
portant'organism  of  the  anaerobic  spore  bearers.  Its  characteristics 
are  the  tetanic  symptoms  produced  by  the  toxin  and  the  strictly  termi- 
nal drum-stick  spores. 

Spores  are  difficult  to  find  in  material  from  wounds  infected  with  tetanus,  but 
readily  develop  in  cultures.  See  notes  under  general  consideration  of  pathogenic 
anaerobes.  Prior  to  the  formation  of  spores  the  organism  is  a  long  thin  bacillus 
(4Xo.4/u).  It  is  motile  and  Gram-positive.  It  liquefies  gelatin  slowly  and  does 
not  coagulate  milk.  The  stab  culture  in  glucose  agar  shows  pine-tree  growth. 
Colonies  on  agar  plates  show  as  fleecy  clouds  and  microscopically  as  felted  filaments. 

Theobald  Smith  recommends  growing  it  in  fermentation  tubes  containing  ordinary 
bouillon,  but  to  which  a  piece  of  the  liver  or  spleen  of  a  rabbit  or  guinea-pig  has  been 


TETANUS  85 

introduced  at  the  junction  of  the  closed  arm  and  the  open  bulb.  By  this  method 
spores  develop  rapidly  in  from  twenty-four  to  thirty-six  hours.  Sporulation  is 
most  rapid  at  37°C.  As  there  is  always  liability  to  postmortem  invasion  of  viscera 
by  ordinary  saprophytes,  Smith  recommends  that  great  care  be  taken  not  to  handle 
the  animal  roughly  in  chloroforming  and  in  pinching  off  pieces  of  the  organ  at 
autopsy.  The  animal  must  be  healthy,  and  the  tubes  to  which  the  piece  of  tissue 
is  added  must  be  proven  sterile  by  incubation.  Smith  calls  attention  to  the  un- 
certainty of  the  temperature  at  which  tetanus  spores  are  killed.  He  shows  that 
some  require  temperature  only  possible  with  an  autoclave.  In  view  of  the  danger 
of  tetanus,  it  is  advisable  to  carefully  autoclave  all  material  going  into  bacterial 
vaccines,  such  as  salt  solution,  bottles  for  holding,  etc. 

Tetanus  seems  to  grow  better  in  symbiosis  with  aerobes;  hence  a 
lacerated  dirty  wound  with  its  probable  contamination  with  various 
cocci,  etc.,  and  its  difficulty  of  sterilization,  offers  a  favorable  soil. 
The  tetanus  bacillus  gives  rise  to  one  of  the  most  powerful  poisons 
known;  it  is  a  soluble  toxin  like  diphtheria  toxin,  and  it  is  estimated 
that  J^joo  grain  is  fatal  for  man. 

There  are  2  toxins,  tetanospasmin  and  tetanolysin,  but  the  former 
is  the  important  one.  Tetanus  toxin  is  twenty  times  as  poisonous  as 
dried  cobra  venom. 

The  antitoxin  is  produced  by  injecting  horses  with  increasing  doses  of  tetanus 
toxin,  following  a  preliminary  dose  of  5000  antitoxin  units.  An  important  point 
is  that  a  horse  used  for  the  production  of  diphtheria  antitoxin  may  become  infected 
with  tetanus  and  his  blood  contain  enough  tetanus  toxin  to  kill.  A  number  of 
children  in  St.  Louis  died  of  tetanus  as  the  result  of  such  an  accident. 

Rosenau  has  established  an  antitoxin  unit  for  tetanus  which  has  the  power  of 
neutralizing  1000  minimal  lethal  doses.  Practically,  it  is  ten  times  the  least 
quantity  of  antitetanic  serum  necessary  to  protect  the  life  of  a  350  grams 
guinea-pig  from  a  test  dose  of  tetanus  toxin  furnished  by  the  hygienic  laboratory. 
(The  necessity  of  some  definite  unit  is  apparent  when  tests  have  shown  that  serum 
stated  to  contain  6,000,000  units  per  c.c.  only  had  a  value  of  90  of  the  official 
American  units.)  Consequently  it  is  a  unit  ten  times  as  neutralizing  as  the  diph- 
theria antitoxin  one.  The  antitoxin  of  tetanus  is  less  efficient  than  that  of  diph- 
theria for  the  following  reasons: 

1.  There  is  about  three  times  as  great  affinity  in  vitro  between  diphtheria  toxin 
and  antitoxin  as  is  the  case  with  tetanus. 

2.  The  tetanus  toxin  has  greater  affinity  for  nerve  cells  than  for  antitoxin. 

3.  Treatment  with  antitoxin  is  successful  after  symptoms  of  diphtheria  appear. 
With  tetanus  it  is  almost  hopeless  after  the  disease  shows  itself.     Hence  the  impor- 
tance of  the  early  bacteriological  examination  of  material  from  a  suspicious  wound 
(rusty  nail). 

4.  The  tetanus  toxin  ascends  by  way  of  the  axis  cylinder,  and  the  antitoxin 
being  in  the  circulating  fluids  cannot  reach  it,  wheras  with  diphtheria  both  toxin 
and  antitoxin  are  in  the  circulation.     Diphtheria  also  selects  the  cells  of  paren- 


86  STUDY  AND   IDENTIFICATION  OF  BACTERIA 

chymatous  and  lymphatic  organs  which  are  more  tolerant  of  injury  than  the  nerve 
cells.  The  dose  of  tetanus  antitoxin  as  a  prophylactic  is  1500  units;  as  a  curative 
agent  5000  to  20,000  units.  Recent  experience  shows  that  it  should  be  injected 
intravenously  when  symptoms  have  manifested  themselves. 

That  the  disease  is  due  to  toxin  is  shown  not  only  experimentally,  but  also  if 
spores  are  carefully  freed  of  all  toxin  by  washing,  and  then  introduced  they  do  not 
cause  tetanus — the  polymorphonuclears  engulfing  them.  The  importance  of  the 
presence  of  ordinary  pus  cocci  in  a  tetanus  wound  may  be  that  the  activity  of  the 
leukocytes  in  phagocytizing  them  allows  the  tetanus  bacillus  to  escape  phagocytosis. 
This  would  also  explain  the  importance  of  necrotic  tissue  in  a  lacerated  wound — 
the  phagocytes  taking  this  up  instead  of  tetanus  bacilli.  The  toxin  is  digested  by 
the  alimentary  canal  juices  and  infection  by  that  atrium  is  improbable.  The  in- 
fection occurs  especially  through  skin  wounds,  and  also  from  those  of  mucous  mem- 
brane. While  tetanus  is  like  diphtheria,  a  disease  in  which  the  bacilli  are  localized 


FIG.  24. — Tetanus  bacilli  showing  end  spores.     (Kolle  and  Wassermann.) 

and  do  not  spread,  yet  recently  Richardson  has  obtained  tetanus  bacilli  in  pure 
culture  from  the  tributary  lymphatic  glands  of  a  "rusty  nail"  wound  of  foot.  The 
cultures  inoculated  into  root  of  tail  of  a  white  rate  caused  the  rat's  death  in  forty- 
eight  hours  with  typical  "seal  gait"  attitude  of  tetanus  in  rats. 

The  usual  period  before  symptoms  occur  is  fifteen  days.  The  shorter  the  period 
of  incubation,  the  more  probably  fatal  the  disease.  The  horse  is  the  most  susceptible 
animal,  next  the  guinea-pig,  then  the  mouse.  Fowls  are  practically  immune. 

In  examining  for  tetanus,  scrape  out  the  granulation  tissue  or  foreign 
material  from  the  suspected  wound  with  a  sterile  Volkmann  spoon  and 
insert  in  a  pocket  made  with  scissors  in  the  subcutaneous  tissues  of  the 
thigh  of  a  guinea-pig.  Animal  inoculation  is  the  practical  method. 
One  may  also  put  some  of  the  suspected  material  in  a  Loffler's  serum 
tube.  Place  in  the  incubator,  under  which  conditions  the  cocci  and 
other  aerobes  grow  luxuriantly  and  enable  the  tetanus  bacillus  to  de- 


THE   GAS  BACILLUS  87 

velop.  From  day  to  day  smell  the  culture,  and  if  an  odor  similar  to  the 
penetrating,  sour,  foul  smell  of  the  stools  of  a  man  who  has  been  on  a 
debauch  be  detected,  it  is  suspicious.  The  nondevelopment  of  a  foul 
odor  is  against  tetanus.  Also  make  smears  from  the  material  and  ex- 
amine for  drum-stick  spores.  If  these  are  found,  heat  the  material 
to  8o°C.  for  one-half  hour,  to  kill  nonsporing  aerobes  and  faculative 
anaerobes,  and  then  inoculate  a  deep  glucose  agar  tube  and  cultivate 
by  Wright's  method.  The  fusiform  lateral  outgrowth  about  the  middle 
of  the  stab  is  characteristic. 

A  more  rapid  method  is  to  draw  up  the  material,  provided  it  be  pus  (tissue  scrap- 
ings may  be  emulsified  in  sterile  salt  solution)  into  a  capillary  bulb  pipette.  Then 
seal  off  the  end  and  heat  the  capillary  bulb  pipette  and  its  contents  in  a  water-bath 
at  8o°C.  for  fifteen  minutes.  Next  break  off  the  sealed  tip  and  stick  the  pipette  into  a 
deep  tube  of  glucose  agar.  When  the  point  reaches  the  bottom,  force  out  the  material 
along  the  line  of  the  stab  as  the  pipette  is  withdrawn.  Cover  the  surface  of  the 
agar  with  sterile  liquid  petrolatum  and  incubate.  Better  anaerobic  conditions 
obtain  where  the  Buchner  or  Wright  method  is  employed. 

Tetanus  produces  no  gas.  Material  for  examination  is  best  obtained  with  a  bulb 
pipette  (containing  a  little  sterile  salt  solution)  which  is  plunged  into  the  agar  and  the 
salt  solution  forced  out  and  drawn  in  where  a  proper  growth  is  noted. 

Spores  form  in  thirty-six  to  forty-eight  hours.  In  injecting  test  animals  it  is 
advisable  to  divide  the  material  to  be  injected  into  two  portions;  one  animal  is 
injected  with  the  material  alone,  the  second  animal  with  tetanus  antitoxin  at  the 
same  time  the  material  is  injected.  Only  the  first  animal  dies  with  tetanic  symptoms. 

B.  Aerogenes  Capsulatus  (Welch,  1891). — This  bacillus  is  apparently 
widely  distributed.  It  is  possibly  the  same  organism  as  Klein's  B. 
enteritidis  sporogenes,  which  is  constantly  present  in  faeces.  It  is  a 
large  capsulated  organism,  which  does  not  form  chains.  Spores  are 
produced  on  blood-serum.  These  are  frequently  absent  on  other 
media.  It  is  questioned  whether  its  pathogenicity  is  other  than  ex- 
ceedingly feeble,  the  presence  of  the  bacillus  in  emphysematous  find- 
ings at  postmortem  being  attributed  to  terminal  or  cadaveric  invasion. 

Cases,  however,  in  the  Philippines,  have  been  reported  following  carabao  horn 
wounds,  in  which  most  serious  and  fatal  results  attended  emphysematous  lesions 
showing  this  bacillus.  The  isolation  of  a  Gram-positive  bacillus  from  a  lacerated 
wound  discharge,  even  in  the  absence  of  emphysema,  is  almost  diagnostic. 

In  milk  cultures  we  have  coagulation  and  from  the  subsequent  development 
of  gas  the  disruption  of  the  coagulum  into  shreds.  An  odor  of  butyric  acid  is 
developed. 

Cultures  in  litmus  milk  show  these  shreds  plastered  against  the  sides  of  the  tube 
and  showing  a  pink  color. 

It  is  the  cause  of  "foamy  organs"  occasionally  present  at  autopsy. 


88 


STUDY   AND   IDENTIFICATION   OF  BACTERIA 


The  best  method  of  diagnosis  is  to  inoculate  the  culture  or  material 

into  the  ear  vein  of  a  rabbit,  kill  it  and 
then  incubate  the  body  at  37°C.  Gas  is 
generated  in  the  organs  in  a  few  hours. 
While  the  organism  is  pathogenic  for 
guinea-pigs  it  has  little  effect  on  rabbits. 
B.  Perfringens. — This  is  the  name  fre- 
quently given  to  the  organism  which  has 
assumed  such  great  importance  on  ac- 
count of  its  causing  the  gas  gangrene  so 
frequently  observed  in  shell  wounds  re- 
ceived in  Belgium.  It  is  considered  to 
be  identical  with  B.  aerogenes  capsulatus 
(Welch's  gas  bacillus)  and  B.  phlegm onis 
empysematosa.  It  is  very  abundant  in 
the  soil  of  highly  fertilized  areas. 

In  size  the  bacillus  is  large,  6X  2.5  microns  with 
square  cut  ends.  It  is  strongly  Gram-positive, 
may  or  may  not  show  a  capsule  and  is  nonmotile. 
When  sporing  the  spore  occurs  toward  one  end 
with  slight  bulging  of  the  rod.  Grown  in  pure 
culture  spores  are  rarely  found,  but  in  symbiosis 
with  staphylococci  they  form  abundantly. 
Cultures  from  the  clothing  of  men  in  the  trenches 
almost  always  show  the  Welch  bacillus  and  less 
frequently  the  tetanus  bacillus.  Streptococci 
were  rather  frequent.  When  a  shell  wound  oc- 
curs we  almost  invariably  have  a  gas  bacillus 
infection  which  during  the  first  few  days  gives 
rise  to  a  foul-smelling  reddish-brown  discharge. 
Smears  from  gas  gangrene  wounds,  showing  such 
discharge,  have  chiefly  the  gas  bacillus  and 
streptococci.  In  the  second  week  the  pus  be- 
comes more  purulent  and  the  gas  bacillus  is  infre- 
quent. Streptococci,  staphylococci  and  coliform 
bacilli  are  abundant.  Glucose  agar  to  which 
about  Ho  c.c.  of  blood  has  been  added  makes 
a  very  favorable  medium  for  the  gas  bacillus. 
It  also  grows  well  on  milk  or  blood-serum. 
Fleming  prefers  neutral  red  egg  medium  for  its 
culturing.  Cultures  of  the  gas  bacillus  from  gas 

gangrene   discharges  when  injected  subcutaneously  into  guinea-pigs  kill   within 
twenty-four  hours  causing  emphysematous  swellings.     Chlorinated  solutions  seem 


FIG.  25. — B.  aerogenes  capsu- 
latus agar  culture  showing  gas 
formation.  (Mac  Neal.) 


THE   GAS  BACILLUS  89 

to  be  more  efficient  against  the  infection  than  the  formerly  recommended  hydrogen 
dioxide. 

Achalme  isolated  a  large  bacillus  from  a  fatal  case  of  rheumatism 
which  is  now  considered  as  having  no  relation  to  acute  rheumatism  and 
which  was  probably  B.  aero  genes  capsulatus. 

Kendall  has  called  attention  to  the  importance  of  this  organism  in  a 
certain  proportion  of  cases  of  summer  diarrhoea  of  infants.  (See  under 
chapter  on  Faeces.) 


CHAPTER  VII 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.    MYCO- 

BACTERIA  AND  CORYNEBACTERIA.    KEY 

AND  NOTES 

Key  for  Bacilli. — Having  branching  characteristics,  as  shown  by  par- 
allelism, branching,  curving  forms,  V-shapes,  clubbing  at  ends,  seg- 
mental  staining,  etc. 

J  Cultures  more  or  less  wrinkled  and  dry. 
Acid-fast.     Mycobactenum.   <  _.       ...  . , 

[  More  like  moulds. 

I.  Grow  rapidly  on  ordinary  media  at  room  temperature. 
Examples:  Timothy  grass  bacillus  of  Moeller  (B.  phlei). 

Mist  bacillus.     Butter  bacilli  as  reported  by  (i)   Rabinowitsch 
and  (2)  Petri. 

II.  Only  grow  at  about  incubator  temperature.  Scanty  growth  or  none  at  all 
on  ordinary  media.  Media  of  preference  are:  (a)  solidified  blood-serum, 
(b)  glycerine  agar,  (c)  glycerine  potato  and  (d)  egg  media. 

1.  Cultures  fairly  moist,  luxuriant,  and  flat.    Op.  temp.  43°C. 
a.  Bacillus  of  avian  tuberculosis. 

2.  Cultures  scanty,  wrinkled,  and  dry.     Appear  in  ten  to  fourteen  days.     Op. 
temp.  38°C.     Bacilli  longer,  narrower,  more  regular  in  outline  and  staining 
than  bovine;  vacuolation  more  marked  (2.5/i).     Smear  from  organs  of  inocu- 
lated guinea-pig  shows  few  bacilli.    Less  virulent  for  rabbits. 

a.  Bacillus  of  human  tuberculosis. 

Cultures  as  above,  but  even  more  scanty.     Bacilli  shorter,  thicker,  less  vacuo- 
lated  (I.SM).     Smear  from  organs  of  guinea-pig  shows  many  bacilli. 

b.  Bovine  tubercle  bacilli. 

3.  Very  difficult  to  cultivate  (Czaplewski). 
Smegma  bacilli  for  various  animals. 

III.  Noncul livable  by  ordinary  methods.  Cultivable  in  symbiosis  with  amoebae. 
(Clegg.)  Duval  cultivated  an  acid-fast  bacillus  on  N.N.N.  medium  con- 
taining i%  glycerine.  Bayon  cultivated  on  placental  juice  glycerine  agar  a 
slightly  acid-fast  diphtheroid  which  changed  to  acid-fast  in  peritoneum  of 
mouse.  Bayon's  organism  thought  to  be  similar  to  Kedrowsky's  diphtheroid 
of  leprosy. 

i.  B.  leprae.     Found  chiefly  in  nasal  mucus  and  in  juice  from  lepra  tubercles. 
Less  often  in  nerve  leprosy. 

90 


NON-PATHOGENIC  ACID-FAST  ORGANISMS 


Nonacid-fast.     Corynebacterium.     TC°lonies  m°r e  flat  and  moist 

(  Like  other  bacteria. 

I.  Do  not  stain  by  Gram's  method. 

i.  B.   mallei    (Glanders).     Characteristic   culture  is   that  on  potato.     Growth 
like  layer  of  honey  by  third  day.     Becomes  darker  in  color,  until  on  eighth 
day  is  reddish  brown  or  opaque  with  greenish-yellow  margin. 
II.  Gram-positive. 

1.  Very  luxuriant  growth  on  ordinary  media.     Colonies  often  yellow  to  brown- 
ish.    B.  pseudodiphtheriae.     Shorter,  thicker  and  stain  uniformly. 

2.  Moderate  growth  on  ordinary  media.     B.  diphtherias.     Best  media  are  blood- 
serum  (Loffler's)  or  glycerine  agar.     Has  metachromatic  granules  at  poles. 

3.  Scanty  and  slow  growth  on  nutrient  media.     B.  xerosis. 

THE  GROUP  OF  ACID-FAST  BRANCHING  BACILLI 

There  is  a  large  and  ever-increasing  number  of  organisms  which 
have  the  same  staining  reactions  as  the  tubercle  bacilli,  but  which  differ 
in  four  important  essentials  of: 

1.  Growing  readily  on  any  media. 

2.  Showing  more  or  less  abundant  growth  or  colonies  in  twenty-four 
hours. 

3.  Having  no  pathogenic  power  for  guinea-pigs  when  inoculated  sub- 
cutaneously. 

4.  Not  requiring  body  temperature  for  development,  but  growing  at 
room  temperature. 

Many  of  these  organisms,  if  injected  intraperitoneally  into  guinea-pigs  will  pro- 
duce a  peritonitis  with  false  membrane.  Some  also  produce  granulation  tissue 
nodules  which  may  be  confused  with  true  tubercles.  For  this  reason  it  is  well  to 
study  the  lesions  in  experimental  tuberculosis  in  the  guinea-pig.  Injected  subcuta- 
neously,  on  either  or  both  sides  of  the  posterior  abdomen  with  the  needle  pointing 
toward  the  inguinal  glands,  we  may  have  caseation  and  ulceration  at  the  site  of 
inoculation.  The  glands  in  relation  enlarge  and  caseate.  Smears  from  these  show 
T.  B.  The  marked  and  characteristic  change  is  the  enormous  enlargement  of  the 
spleen,  which  is  studded  with  grayish  and  yellow  tubercles.  Make  smears  and  cul- 
tures from  the  spleen.  The  death  of  the  guinea-pig  usually  occurs  in  about  two 
months.  The  lesions  may  be  looked  for  at  three  to  five  weeks. 

These  nonpathogenic  acid-fast  bacilli  are.  of  greatest  importance  by  reason  of 
their  possible  confusion  with  the  true  tubercle  bacilli.  Their  colonies  correspond 
more  or  less  with  different  types  of  tubercle  bacilli  colonies,  being  either  dry  and 
wrinkled  like  human,  or  moist  and  irregularly  flat  as  avian.  Eventually  the  moist 
colonies  become  dry  and  wrinkled.  They  have  been  isolated  from: 

1.  Butter  and  milk. 

2.  From  grasses,  especially  in  timothy  grass  infusion. 

3.  In  various  excretions  of  animals,  as  in  dung,  urine,  etc. 

4.  Normally  in  man — from  skin,  nasal  mucus,  cerumen,  and  tonsillar  exudate. 


STUDY   AND    IDENTIFICATION   OF  BACTERIA 


It  is  important  to  remember  that  such  organisms 
have  very  rarely  been  reported  from  pulmonary  le- 
sions, and  when  present  they  have  been  considered 
as  probably  causative. 

The  present  view  is  that  the  finding  of  tubercle 
bacilli  in  sputum  has  practically  as  great  value  as  it 
had  before  we  knew  of  these  various  acid-fast  bacteria. 

Tubercle  Bacillus  (Koch,  1882).— This  is 
a  rather  long,  narrow  rod,  3X0.3^1.  In  the 
human  type  it  tends  to  show  a  beaded  ap- 
pearance, this  not  being  due  to  spores,  how* 
ever.  In  the  bovine  type  the  staining  is  more 
solid,  the  organism  shorter  and  thicker,  and 
shows  even  a  more  scanty  growth  than  human 
T.  B.  It  has  been  established  that  many  of 
the  tuberculous  affections  of  man,  especially 
those  of  the  skin,  bone,  and  mesenteric  glands, 
.are  of  the  bovine  type,  while,  as  a  rule,  pul- 
monary and  laryngeal  lesions  are  of  the  hu- 
man type.  Experiments  by  various  com- 
missions in  different  countries  have  shown 
that  human  and  bovine  types  are  very  closely 
related  and  that  not  only  may  a  bovine  strain 
affect  man,  but  that  human  T.  B.  may  infect 
young  calves.  As  bacilli  of  the  bovine  type 
have  frequently  been  reported  in  intestinal 
and  mesenteric  tuberculosis  of  children  it 
shows  the  importance  of  sterilizing  cows' 
milk.  Koch  considered  human  infection  from 
bovine  sources  as  of  very  rare  occurrence. 

Although  Kossel  has  found  only  two  cases  of 
bovine  T.  B.  in  709  cases  of  pulmonary  tuberculosis 
yet  for  the  other  types  the  findings  are  different. 
Leaving  out  of  consideration  the  frequency  of  in- 
fections with  bovine  T.  B.  in  children,  recent  sta- 
tistics have  shown  that  in  adults  about  4%  of  cervi- 
cal adenitis,  22%  of  tabes  mesenterica  and  3. 5%  of 
bone  and  joint  tuberculosis  are  due  to  bovine  strains  of  T.  B. 

Park  and  Krumweide,  in  a  study  of  more  than  1000  cases,  found 
about  10%  due  to  bovine  tuberculosis.     Of  686  adult  cases  only  1.3% 


FIG.  26. — Bacillus  tuber- 
culosis; glycerine  agar-agar 
culture,  several  months  old. 
(Curtis.) 


TYPES  OF  TUBERCLE  BACILLI  93 

showed  bovine  strains  while  352  cases  under  sixteen  showed  approxi- 
mately 25%  of  bovine  infection.  Of  592  cases  of  pulmonary  tuberculosis, 
in  children  and  adults,  not  a  single  case  could  surely  be  regarded  as 
bovine. 

A  subject  of  great  moment  is  that  of  the  atrium  of  infection  in  tuberculosis. 
While  75%  or  more  of  human  cases  are  of  the  respiratory  tract,  yet  we  now  have 
views  that  Cornet's  idea  of  T.  B.  containing  dust  being  aspirated,  or  Fliigge's  spray 
method  of  infection  from  droplets  of  sputum  in  coughing,  may  be  but  rarely  operative. 
The  path  from  intestinal  tract  to  thoracic  duct  and  lungs  is  direct,  so  that  tuber- 
culous bronchial  glands  of  lung  infection  may  be  by  way  of  intestines.  Some 
European  statistics,  using  von  Pirquet's  method,  have  shown  90%  of  children 
under  fourteen  infected  while  similar  American  ones  have  shown  about  50%.  The 
prevailing  idea  is  that  we  get  our  infection  in  childhood  and  show  the  disease  as  we 
approach  adult  life.  Infection  after  adult  life  is  exceedingly  rare,  as  shown  by  the 
rarity  of  the  disease  in  attendants  at  institutions  for  the  tuberculous.  The 
respiratory  atria  would  be  just  as  operative  for  adults  as  children  so  the  probable 
explanation  is  that  children  in  crawling  about,  where  tuberculous  material  is 
accessible,  may  contaminate  the  fingers  and  have  infection  take  place  by  the  digestive 
tract.  With  bovine  infections  we  are  sure  that  infection  of  man  takes  place  almost 
exclusively  by  the  alimentary  tract. 

The  British  Royal  Commission  in  its  final  report  of  July,  1911,  considered  three 
types  of  T.  B. 

I.  The  bovine  type  belonging  to  the  natural  tuberculosis  of  cattle. 
II.  The  human  type.     The  type  more  generally  found  in  man. 

III.  The  avian  type,  belonging  to  natural  tuberculosis  of  fowls. 

The  bovine  type  grows  slowly  on  serum  and  at  the  end  of  two  to  three  weeks 
shows  only  a  thin  grayish  uniform  growth  which  is  not  wrinkled  and  not  pigmented. 
The  human  type  grows  more  rapidly  and  tends  to  become  wrinkled  and  pigmented. 
Subcutaneous  inoculation  of  50  mg.  of  culture  into  the  neck  of  calves  produced 
generalized  tuberculosis.  A  similar  injection  of  human  T.  B.  does  not  cause  general- 
ized tuberculosis  but  only  an  encapsulated  local  lesion. 

Intravenous  injection  of  o.oi  to  o.i  mg.  of  bovine  T.  B.  into  rabbits  causes  general 
miliary  tuberculosis  and  death  within  five  weeks.  With  human  T.  B.  in  doses  of 
o.i  to  i.o  mg.,  similarly  injected,  the  majority  of  rabbits  live  for  three  months. 

Subcutaneous  injection  of  10  mg.  bovine  T.  B.  causes  death  in  28  to  101  days. 
Similar  injection  of  human  T.  B.  in  doses  up  to  100  mg.  did  not  kill  the  rabbits  after 
periods  of  from  94  to  725  days.  The  duration  of  life  in  injected  guinea-pigs  is 
longer  with  human  than  with  bovine  T.  B. 

Subcutaneous  injections  of  bovine  T.  B.  into  cats  produces  generalized  tubercu- 
losis while  the  cat  is  resistant  to  human  T.  B.  thus  given. 

Recent  statistics  (Beitzke)  show  tuberculous  lesions  in  58%  of  adults 
at  autopsy — Naegli's  figures  were  about  90%. 

It  is  a  question  whether  the  avian  type  is  absolutely  distinct;  many 
experiments  having  indicated  the  impossibility  of  infecting  fowls  with 


94  STUDY  AND   IDENTIFICATION  OF  BACTERIA 

human  T.  B.  Nocard,  by  inserting  collodion  sacs  containing  bouillon 
suspensions  of  human  T.  B.,  claims  to  have  changed  these  to  the  avian 
type.  The  avian  type  grows  at  43°C.  fairly  luxuriantly,  as  a  moist, 
more  or  less  spreading  culture.  It  grows  much  better  on  glycerinated 
agar  than  on  serum.  Morphologically  they  are  like  the  human  type, 
but  show  less  tendency  to  form  compact  masses.  Very  pleomorphic. 
Have  been  reported  from  sputum  of  man  (doubtful). 

Fowls  become  infected  by  intravenous  or  subcutaneous  injection  or  as  the  result 
of  feeding.  After  feeding  the  lesions  are  chiefly  of  the  alimentary  tract;  after  in- 
jections, of  spleen,  liver  and  lungs.  Avian  T.  B.  is  more  virulent  for  rabbits  than 
human  T.  B.  but  less  so  than  bovine  T.  B.  The  mouse  is  the  only  animal  besides  the 
rabbit  in  which  avain  T.  B.  can  cause  a  generalized  tuberculosis.  The  conclusions 
are  that  there  is  no  danger  to  man  from  avian  T.  B.  With  the  bovine  type  it  is 
quite  different  as  nearly  one-half  of  the  deaths  in  young  children  from  abdominal 
tuberculosis  were  due  to  bovine  T.  B.  and  to  that  type  alone.  Not  only  in  children, 
but  in  adolescents  suffering  from  cervical  gland  tuberculosis,  a  large  proportion  were 
caused  by  bovine  types.  The  bovine  type  is  also  an  important  factor  in  lupus. 

There  is  also  a  fish  tuberculosis.  This  organism  grows  much  more 
rapidly  than  the  other  types  (three  to  four  days),  and  grows  best  at 
24°C.,  growth  ceasing  at  36°C.  The  colonies  are  round  and  moist. 

It  is  certain  that  many  of  the  symptoms  usually  noted  in  the  tuberculous  are  due 
to  secondary  infections.  Pettit,  by  careful  blood  cultures,  obtained  the  Pneumo- 
coccus  in  24  cases  and  the  Streptococcus  in  36  cases  out  of  130  cases  studied.  He 
used  from  5  to  20  c.c.  of  blood  from  the  vein.  Positive  blood  cultures  were  obtained 
in  68%  of  far-advanced  cases,  45%  of  advanced  cases  and  16%  of  incipient  cases. 

The  best  culture  medium  for  primary  cultures  is  blood-serum  or, 
better,  a  mixture  of  yolk  of  egg  and  glycerine  agar.  Petroff 's  medium 
and  Dorsett's  egg  medium  are  also  used.  In  subcultures,  either  glycer- 
ine agar,  glycerine  potato,  or  glycerine  bouillon  make  good  media. 

In  inoculating  media  from  tuberculous  material,  as,  say,  from  a  tuberculous  gland 
or,  more  practically,  from  the  spleen  of  a  guinea-pig,  the  material  must  be  thoroughly 
disintegrated  or  rubbed  on  the  surface  of  the  media  so  that  individual  bacilli  may 
rest  on  the  surface  of  the  culture  media.  In  growing  in  flasks  in  glycerine  bouillon 
a  surface  growth  is  desired.  The  cylindrical  flask  of  Koch  gives  a  better  support 
to  the  pellicle  than  an  Erlenmeyer  one.  In  inoculating,  a  scale  of  such  a  surface 
growth  or  a  grain  from  the  growth  on  a  slant  should  be  deposited  on  the  surface  of 
the  glycerine  bouillon  in  the  flask.  In  cultivating  from  sputum  use  Petroff's 
medium. 

Inasmuch  as  the  filtrate  from  cultures  has  little  toxic  effect,  the 
poison  is  assumed  to  be  intracellular. 


DIAGNOSTIC  TESTS  FOR  TUBERCULOSIS  95 

Koch's  "Old  Tuberculin,"  which  was  simply  a  concentrated  5%  glycerine  bouillon 
culture,  is  now  principally  used  in  diagnosis.  It  was  prepared  as  follows: 

After  four  to  six  weeks  the  surface  growth  begins  to  sink  to  the  bottom  of  the 
flask.  This  fully  developed  culture  is  evaporated  over  a  water-bath  at  8o°C.  to 
one-tenth  the  original  volume.  It  is  then  filtered,  the  final  product  containing 
about  40%  of  glycerine. 

Koch's  "New  Tuberculin"  or  tuberculin  "R"  was  introduced  in  1897.  In  this, 
virulent  bacilli  are  dried  in  vacua  and  ground  up  until  stained  smears  fail  to  show 
intact  bacilli.  One  gram  of  such  material  is  triturated  with  100  c.c.  water  and 
centrifugalized.  The  supernatant  fluid  is  removed  and  is  designated  "T.  O." 
The  residue  is  then  dried,  triturated  in  water  and  centrifugalized.  Subsequent 
trituration  and  centrifugalization,  preserving  each  time  the  supernatant  suspension, 
gives  the  new  tuberculin.  It  has  been  found  at  times  to  contain  virulent  T.  B. 

Koch's  bazillen  emulsion  has  been  more  recently  introduced  by  Koch  (1901). 

This  is  simply  a  suspension  of  ground-up  bacilli  in  50%  glycerine  solution. 

It  really  is  "T.  O"  and  "T.  R"  combined  and  contains  5  mg.  of  bacillary 
substance  in  i  c.c.  Another  preparation  is  the  bouillon  filtrate  of  Denys.  This  is 
the  unheated  Chamberland  filtrate  of  broth  cultures  of  human  T.  B.  It  contains 
Y±%  phenol. 

In  the  use  of  T.  R.  and  of  bazillen  emulsion,  Sir  A.  Wright  recommends  doses 
of  Kooo  nig.,  and  he  rarely  goes  beyond  Kooo  rng.  in  treatment.  These  prod- 
ucts come  in  i  c.c.  bottles  containing  5  mg.  of  bacillary  material.  It  is  convenient 
to  remove  %o  c-c.,  containing  i  mg.  Add  this  to  10  c.c.  of  glycerine  salt  solution 
with  %%  of  lysol.  Each  c.c.  contains  Ko  nig.  One  c.c.  of  this  stock  solution 
added  to  99  c.c.  of  salt  solution,  with  Y±%  of  lysol,  would  give  a  working  solution, 
each  c.c.  of  which  would  contain  Mo  00  mS-  of  tuberculin. 

For  diagnostic  tuberculin  reactions  we  have  the  following: 

1.  Subcutaneous  injection  of  J£  mg.     If  no  reaction  occurs  in  four 
or  five  days  we  may  increase  to  i  to  5  mg. 

Positive  reactions  show  (a)  constitutional  symptoms  of  fever,  malaise  and  possibly 
chill;  (6)  focal  symptoms,  as  when  a  tuberculous  gland,  joint  or  skin  involvement 
becomes  active,  and  (c)  local  reaction  as  shown  by  the  tenderness,  induration  or 
inflammation  at  the  site  of  injection. 

2.  Variations  in  opsonic  index. 

3.  Instillation  into  one  eye  of  a  drop  of  J£%  or  i  %  solution  of  puri- 
fied tuberculin. 

Reaction  is  shown  by  redness,  especially  of  inner  canthus,  in  twelve  to  twenty- 
four  hours  (Calmette).  A  previous  instillation  may  sensitize  a  nontuberculous  case 
and  a  second  application  of  the  drop  may  give  an  erroneous  diagnosis.  This  test 
should  not  be  used  in  persons  over  fifty  or  when  there  is  any  disease  of  the  eye  to  be 
used  or  for  that  matter  of  the  other  eye.  For  instance,  in  corneal  opacities,  due  to 
T.  B.  keratitis,  a  focal  reaction  would  occur. 


96  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

4.  The  cutaneous  inoculation  method  (similar  to  ordinary  vaccina- 
tion methods).     Scarify  two  small  areas  on  the  arm  (J^o  mcn  m  diame- 
ter), about  2  inches  apart.     Rub  in  one  a  drop  of  old  tuberculin,  in 
the  other  a  drop  of  25%  tuberculin.     As  a  control  scarify  a  spot  mid- 
way and  to  one  side  of  the  others  and  rub  in  i  drop  of  0.5%  carbolic 
glycerine.     The  appearance  of  bright  red  papules  in  twenty-four  hours 
indicates  reaction  (von  Pirquet). 

It  is  now  recommended  to  deposit  a  loopful  of  undiluted  tuberculin  on  the  skin 
and  below  that  a  loopful  of  saline.  A  linear  incision  with  a  sharp  scalpel  or  glass 
needle  is  made  through  the  saline  and  then  through  the  drop  of  tuberculin  trying  not 
to  draw  blood.  After  two  to  five  minutes  the  tuberculin  is  wiped  off.  No  dressing  is 
required.  Reaction  usually  appears  as  induration  or  inflammatory  areola  or  vesicles 
after  twenty-four  hours,  but  may  be  delayed  forty-eight  hours.  This  is  the  method 
of  preference. 

5.  Intracutaneous  inoculation  of  i  drop  of  a  i-iooo,  i-ioo  or  i-io 
dilution  of  old  tuberculin  (Mantoux  and  Moussu). 

Webb  recommends  hypodermic  needle  points  which  have  been  dipped  in  old 
tuberculin  and  the  points  allowed  to  dry.  A  drop  of  water  is  placed  on  the  skin 
and  the  needle  points  having  been  moistened  in  it  are  plunged  through  the  skin  and 
withdrawn  with  a  twist.  A  definite  lump  shows  a  positive  reaction. 

6.  Ointment  tuberculin  test.     Rub  in  50%  ointment  of  tuberculin 
in  lanolin.     Reaction  is  shown  by  dermatitis  with  reddened  papules 
in  twenty-four  to  forty-eight  hours  (Moro). 

7.  Inoculation  of  bovine  and  human  tuberculin  to  diagnose  type  of 
infection  (Detre).     Of  questionable  value. 

Ebright  injects  the  suspected  material  into  the  subcutaneous  tissue  of  one  side 
of  the  abdomen  of  3  guinea-pigs.  At  the  end  of  one  week  an  injection  into  the 
other  side  of  the  abdomen  of  one  of  the  guinea-pigs  of  H  c.c.  tuberculin  is  given. 
Twenty-four  hours  later  smears  are  made  from  the  original  site  of  inoculation  and 
examined  for  tubercle  bacilli.  If  negative  this  is  repeated  with  a  second  guinea- 
pig  at  the  end  of  the  second  week  and  finally  at  the  end  of  the  third  week  with  the 
third  guinea-pig. 

Bloch's  method  is  to  damage  the  lymphatic  glands  in  the  inguinal  region  by 
squeezing  the  tissue  between  the  fingers.  Injections  made  there  of  tuberculosis 
material  show  abundant  tubercle  bacilli  in  these  damaged  glands  in  ten  to  twelve 
days. 

In  staining  it  is  better  to  use  the  Ziehl-Neelsen  method,  decolorizing 
with  3%  hydrochloric  acid  in  95%  alcohol.  The  alcohol,  for  all  prac- 
tical purposes,  enables  us  to  eliminate  the  smegma  and  similar  bacilli, 
these  being  decolorized  by  such  treatment.  There  are  two  objections 


LEPROSY  97 

| 

to  the  Gabbett  method,  where  decolorizer  and  counterstain  are  com- 
bined: i.  We  cannot  judge  of  the  degree  of  decolorization  — we  are 
working  in  the  dark;  and  2.  the  matter  of  elimination  of  smegma  bacilli 
is  impossible. 

Pappenheim's  method,  in  which  corallin  and  methylene  blue  are  dissolved  in 
alcohol,  does  not  appear  to  have  an  advantage  over  acid  alcohol.  As  a  practical 
point  when  the  question  of  tuberculosis  of  the  genito-urinary  tract  is  involved, 
inoculate  a  guinea-pig  with  urinary  sediment. 

It  must  be  remembered  that  in  young  cultures  of  tubercle  bacilli  many  of  the 
rods  are  nonacid-fast,  taking  the  blue  of  the  counter  stain,  while  older  rods  are 
acid-fast.  This  frequently  causes  suspicion  of  a  contaminated  culture. 

Discussion  has  arisen  as  to  the  granules  of  Much.  These  are  considered  by  Much 
as  resistant  forms  while  others  consider  them  degeneration  forms  of  tubercle  bacilli. 
At  any  rate  material  containing  only  these  Gram-positive  granules  and  no  acid-fast 
rods  may  when  injected  into  animals  give  rise  to  tuberculosis  and  acid-fast  bacilli. 

The  combination  of  the  acid-fast  and  Gram-staining  methods  as  recommended 
by  Fontes  is  very  satisfactory. 

Bacillus  Leprae  (Hansen,  1874). — This  is  the  cause  of  leprosy.  In 
nodular  leprosy  the  organism  is  readily  and  in  the  greatest  abundance 
found  in  the  juice  of  the  tubercles  of  the  skin,  and  secretions  of  ulcera- 
tions  of  nasal  and  pharyngeal  mucosa. 

The  earliest  lesion  is  probably  a  nasal  ulcer  at  the  junction  of  the  bony  and 
cartilaginous  septum.  Scrapings  from  this  ulcer  may  give  an  early  diagnosis. 

In  the  skin  they  are  chiefly  found  in  the  derma  packed  in  the  so-called  lepra 
cells.  The  process  is  granulomatous  but  does  not  show  the  caseation  of  tubercu- 
losis or  the  predominant  plasma  cells  of  syphilis.  The  bacilli  are  also  found  engulfed 
in  the  endothelial  cells  lining  the  lymphatics. 

They  are  also  found  in  the  glands  in  relation  to  the  superficial  lesions.  The 
bacilli  are  found  in  smaller  numbers  in  the  liver  and  spleen.  In  anaesthetic  or  nerve 
leprosy  they  are  found  in  small  numbers  in  the  granuloma  tissue  which  affects  the 
interstitial  connective  tissue  of  the  peripheral  nerves.  Also,  rarely,  in  the  anaesthetic 
spots  of  nerve  leprosy. 

The  leprosy  bacilli  are  found  in  profusion  in  the  granulomatous  tissue  of  the 
corium  and  subcutaneous  structures  of  the  leprous  nodules,  chiefly  within  cells 
called  "lepra  cells"  and  also  within  endothelial  and  connective-tissue  cells  as  well 
as  lying  free,  packed  in  lymphatic  channels,  the  so-called  "globi." 

The  leprosy  bacillus  may  be  distinguished  from  the  tubercle  bacillus 
by  the  following  points: 

i.  The  presence  ordinarily  of  huge  numbers  of  bacilli  often  grouped 
in  packets  like  a  bundle  of  cigars  tied  together.  It  will  be  remembered 
that  it  is  very  difficult  to  find  even  a  single  tubercle  bacillus  in  a  skin 
lesion.  Leprosy  bacilli  form  palisade  groups  but  not  chains. 


98  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

•#• 

2.  The  leprosy  bacilli  stain  more  solidly  and  when  granules  are  pres- 
ent they  are  coarser  and  more  widely  separated  than  the  fine  granula- 
tions of  the  tubercle  bacillus. 

3.  They  do  not  stand  decolorization  quite  as  well  as  the  tubercle 
bacillus.     With  20%  sulphuric  acid  in  water  they  hold  their  color  almost 
as  well  as  tubercle  bacilli  but  with  3%  HCL  in  alcohol  they  decolorize 
in  about  two  hours  as  against  twelve  to  twenty-four  hours  for  the 
tubercle  bacillus. 

4.  Leprosy  bacilli  have  neither  been  surely  cultivated  nor  surely 
inoculated  with  pathogenic  results  into  guinea-pigs  or  other  experi- 
mental animals  and  it  is  by  the  negative  results  upon  cultivating  or 
animal  inoculation  that  we  have  our  surest  method  of  differentiation 
from  tubercle  bacilli. 

Leprosy  bacilli  are  chiefly  spread  through  the  lymphatics,  but  in  nodular  leprosy, 
their  occurrence  in  the  blood  stream  during  the  febrile  accessions  is  so  constant  that 
this  route  may  also  be  of  importance.  Next  to  the  corium  they  are  most  abundant 
in  the  lymphatic  glands.  They  stain  readily  by  Gram's  method. 

A  great  amount  of  work  has  been  done  within  recent  years  in  attempting  to 
cultivate  the  leprosy  bacillus. 

In  1900  Kedrowsky,  culturing  material  from  3  cases  of  leprosy  obtained  diph- 
theroids  from  2  and  a  streptothrix  from  i.  A  rabbit  was  inoculated  first  in- 
travenously and  later  intraperitoneally  with  this  nonacid-fast  streptothrix  and 
when  killed  six  months  later  showed  peritoneal  nodules  from  which  both  diphtheroids 
and  acid-fast  bacilli,  but  not  a  streptothrix,  were  recovered  culturally.  Injection  of 
cultures  of  the  acid-fast  bacilli  and  diphtheroids  into  rabbits  and  mice  produced 
nodules  which,  when  cultured,  showed  acid-fast  organism  or  diphtheroids.  In 
1901,  he  cultivated  a  diphtheroid  from  a  fourth  case  of  leprosy. 

Fraser  and  Fletcher  working  with  Kedrowsky's  culture  produced  peritoneal 
nodules  with  the  killed  as  well  as  the  living  organism.  They  were  able  to  produce 
the  same  results  with  B .  phlei. 

With  emulsions  of  leprous  nodules,  rich  in  leprosy  bacilli,  they  could  not  produce 
similar  lesions  in  the  experimental  guinea-pigs. 

Rost  obtained  a  culture  on  a  salt-free  medium  from  which  he  prepared  his  leprolin 
by  a  process  similar  to  that  used  for  old  tuberculin.  It  was  claimed  that  leprolin 
had  marked  curvative  power  in  leprosy.  Recently  Williams  and  Rost  have  culti- 
vated a  streptothrix  on  a  medium  containing  milk. 

Clegg,  by  inoculating  his  medium  with  cultural  amoebae,  obtained  growth  of 
diphtheroid  organism,  with  acid-fast  tendencies,  from  the  spleen  pulp  of  lepers. 

Duval,  by  using  media  containing  amino-acids,  as  result  of  tryptic  digestion, 
brought  forward  two  organisms,  one  of  which  was  a  diphtheroid  and  grew  luxuriantly 
while  the  other  showed  a  slow  scanty  growth  and  was  acid-fast. 

Bayon,  by  using  placental  media,  isolated  an  organism  rather  resembling  that 
of  Kedrowsky.  These  organisms  alone  responded  to  immunity  tests  when  si 


DIAGNOSIS  OF  LEPROSY  99 

were  made  by  Bayon  and  they  alone  gave  rise  to  tissue  changes  resembling  those  of 
leprosy  when  injected  into  animals. 

Professor  Deycke  obtained  a  streptothrix-like  growth  from  the  granulomatous 
tissue  of  excised  leprous  nodules.  The  ethereal  extract  from  this  culture  gave  a 
neutral  fat  which  he  called  nastin  and  which  is  the  basis  of  a  leprosy  treatment. 

Quite  recently  and  after  working  for  eighteen  months,  with  material 
from  32  nonulcerative  cases  of  nodular  leprosy,  not  only  with  media  as 
recommended  by  Duval,  Rost  and  Bayon,  but  with  blood  and  serum 
culture  media,  both  by  aerobic  and  anaerobic  procedures,  Fraser  has 
been  unable,  in  a  single  instance,  to  obtain  any  evidence  of  growth  from 
this  wealth  of  leprosy  material. 

As  being  opposed  to  the  possibility  of  culturing  the  human  leprosy  bacillus,  it 
may  be  stated  that  most  of  the  experiments  along  this  line  with  rat  .leprosy,  a 
disease  occurring  naturally  in  rats  and  caused  by  an  organism  almost  identical,  as 
to  lesions  produced,  with  the  leprosy  bacillus,  have  been  negative.  Bayon,  however, 
states  that  he  has  cultivated  the  bacillus  of  rat  leprosy. 

Recently  a  leprosy-like  disease  of  rats  has  been  reported  in  which  there  are  two 
types:  i.  A  skin  affection  and  2.  a  glandular  one.  In  this  disease,  acid-fast, 
bacilli,  alike  in  all  respects  to  leprosy  bacilli,  have  been  found.  Deane  has  obtained 
a  diphtheroid-like  organism  in  culture,  which  is  nonacid-fast.  This  same  finding 
has  been  obtained  in  cultures  considered  positive  in  human  leprosy. 

There  have  been  many  reports  of  positive  findings  with  the  Wassermann  test  in 
cases  of  tubercular  leprosy  but  such  reports  are  considered  doubtful  by  many. 
Butler,  in  the  Philippines,  has  found  that  the  lepers  gave  no  higher  percentage  of 
positive  Wassermann  reactions  than  did  the  nonleprous  native  patients  at  his 
clinic. 

There  is  nothing  definitely  known  as  to  method  of  transmission  of 
the  disease. 

In  rat  leprosy  it  has  been  found  that  infection  of  other  rats  takes  place  as  readily 
through  slight  abrasions  of  the  skin  as  when  material  is  injected  subcutaneously. 

The  idea  is  that  natural  infection  occurs  by  way  of  the  skin  and  through  the 
lymphatics.  There  is  no  evidence  that  insects  play  a  part  in  transmission. 

Rat  leprosy  prevails  extensively  in  Europe,  Asia  and  America.  Although  similar 
etiologically  and  pathologically  there  does  not  seem  to  be  any  connection  between 
the  disease  in  rat  and  in  man,  as  is  the  case  with  human  and  rat  plague. 

Laboratory  Diagnosis,— The  usual  procedure  is  to  scrape  a  spot  or 
nodule  with  a  scalpel  until  the  epidermis  has  been  gone  through  and 
then  smear  out  the  serous  exudate  on  a  slide  and  stain  by  the  Ziehl- 
Neelsen  acid-fast  method  or  by  Gram's  stain.  Twenty  percent  sul- 
phuric acid  is  less  apt  to  decolorize  than  the  3%  acid  alcohol,  the  lep- 
rosy bacilli  being  less  resistant  to  acid  alcohol  decolorization  than  to 


100  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

aqueous  acid  solutions.  There  is  a  great  variation  in  the  resistance  to 
decolorization  of  leprosy  bacilli,  a  preparation  from  one  case  holding  its 
color  almost  as  well  as  tubercle  bacilli,  while  material  from  another 
case  may  decolorize  very  easily. 

ik 

I  am  partial  to  Tschernogabow's  technic.  In  this  one  punctures  the  sub- 
epithelial  granulomatous  tissue  with  a  capillary  pipette,  the  end  of  which  has  been 
broken  off  by  tapping  the  point  in  order  to  give  a  cutting  point,  and  the  serum  which 
exudes  is  smeared  out  and  stained. 

Some  prefer  emulsifying  a  piece  of  the  tissue  and  centrifuging  and  staining  the 
sediment.  Quite  recently  the  antiformin  method  of  treating  leprous  tissue,  as  for 
tuberculous  tissue,  has  been  used. 

Many  insist  that  the  best  method  is  to  cut  out  small  sections  of  the  lesion,  going 
well  into  normal  tissue,  and  putting  through  paraffin  and  cutting  thin  sections  and 
staining.  Gram's  method,  counterstaining  with  Bismark  brown  gives  beautiful 
preparations.  For  acid-fast  staining  first  stain  with  haematoxylin  to  obtain  a  his- 
tological  background  and  then  steam  with  carbol-fuchsin,  decolorize  very  briefly 
with  acid  alcohol,  then  through  absolute  alcohol  and  xylol. 

Of  the  greatest  diagnostic  value  is  the  staining  of  the  nasal  mucus 
or  scrapings  from  ulcerations  on  nasal  septum  for  leprosy  bacilli. 
These  are  often  found  in  the  characteristic  cigar  package  bundles  or 
engulfed  in  lepra  cells.  A  standard  procedure  is  to  give  60  grains  of 
iodide  of  potash  to  cause  a  drug  coryza,  in  the  secretions  of  which  lep- 
rosy bacilli  may  be  found.  However,  one  will  have  better  success  if 
the  nasal  secretion  be  obtained  at  a  time  when  a  natural  coryza  exists. 

Thibault  examined  the  nasal  mucus,  gland  juice  and  blood  of  30  lepers.  He 
obtained  leprosy  bacilli  in  the  nasal  mucus  of  20,  in  the  gland  puncture  juice  of  18, 
and  in  the  blood  of  7. 

Hollman  detected  leprosy  bacilli  in  the  nasal  mucus  of  90%  of  58  nodular 
cases,  of  67%  of  6  mixed  leprosy  and  of  45%  of  anaesthetic  cases,  after  making  329 
examinations. 

Leprosy  bacilli  are  apt  to  be  found  in  the  blood  of  nodular  cases,  especially  at  the 
time  of  the  febrile  accessions.  The  blood  is  best  taken  in  5  or  10  c.c.  quantities  into 
i%  sodium  citrate  in  distilled  water.  After  centrifuging,  the  sediment  is  treated 
with  10%  antiformin,  at  37°C.  for  one  hour.  Again  centrifuging,  and  washing,  the 
sediment  is  smeared  out  on  a  slide  and  stained.  The  bacilli  are  not  apt  to  be  found 
in  the  blood  of  cases  of  nerve  leprosy. 

Gland  puncture  has  recently  been  considered  as  an  important  diagnostic  procedure 
in  leprosy. 

It  must  not  be  forgotten  that  while  the  finding  of  leprosy  bacilli  is 
usually  very  easy  in  the  nodules  of  nodular  leprosy  it  is  a  painstaking 
and  discouraging  procedure  with  the  spots  of  nerve  leprosy.  Even  the 


GLANDERS  IOI 

affected  nerves,  at  autopsy,  often  fail  to  show  bacilli.     For  nerve-lep- 
rosy the  examination  of  nasal  mucus  is  of  prime  importance. 

The  X-ray  has  been  utilized  in  the  recognition  of  the  very  early,  trophic  changes  in 
bone,  showing  the  commencing  absorption  of  phalanges. 

NONACID-FAST    BRANCHING    BACILLI 

Bacillus  Mallei  (Loffler  and  Shutz,  1882).— This  is  the  cause  of  a 
rather  common  disease  of  horses.  When  affecting  the  superficial 
lymphatic  glands,  it  is  termed  "farcy;"  when  producing  ulceration  of 
nasal  mucous  membrane,  the  term  "glanders"  is  used. 

In  man  there  are  2  types  of  glanders — chronic  and  acute.  In  the 
chronic  form  an  abrasion  becomes  infected  from  contact  with  glanders 
material  and  an  intractable  foul  discharging  ulceration  results.  This 
may  persist  for  months  with  lymphatic  involvement  or  may  become 
acute.  The  acute  form  may  also  develop  from  the  start  and  the  cases 
are  usually  diagnosed  as  pyaemia.  There  is  great  prostration  with 
marked  pains  of  the  extremities.  Death  invariably  results  in  acute 
glanders.  The  bacillus  is  a  narrow,  slightly  curved  rod,  about  3X0.3^1. 
It  is  nonmotile  and  Gram-negative.  It  at  times  presents  a  beaded  ap- 
pearance. In  subculture  on  agar  or  blood-serum  the  growth  is  some- 
what like  typhoid  but  more  translucent.  In  original  cultures  from  pus 
or  tissues  the  colonies  may  not  show  themselves  for  forty-eight  hours. 

As  the  organism  does  not  tend  to  invade  the  blood  stream,  blood 
cultures  are  apt  to  be  negative.  The  glanders  bacillus  grows  best  on 
an  acid  glycerine  agar  (+2). 

The  characterisitc  culture  is  that  on  potato.  Grown  at  37°C.,  we 
have  a  light  brown  or  yellowish  honey-like  or  mucilaginous  growth, 
which  by  the  end  of  a  week  spreads  out  and  takes  a  cuprous  oxide  like 
reddish  tint  with  greenish  borders.  The  potato  assumes  a  dirty  brown 
color.  This  and  the  inoculation  of  a  guinea-pig  are  the  chief  diagnostic 
measures.  If  the  material  is  injected  intraperitoneally  into  a  male 
guinea-pig,  marked  swelling  of  the  testicles  is  noted  within  forty-eight 
hours,  at  the  earliest,  to  seven  to  ten  days.  Cultures  should  be  made 
from  this  swollen  testicle  as  other  organisms  than  glanders  may  bring 
it  about. 

Only  the  B.  pyrocyaneus  and  cholera  vibrios  give  a  similar  coloration  of  potato 
These  organisms,  however,  are  easily  differentiated.  The  glanders  bacillus  is  the 
most  dangerous  of  laboratory  cultures  and  should  be  handled  with  extreme  care. 


102 


STUDY   AND   IDENTIFICATION   OF  BACTERIA 


The  best  stains  are  carbol  thionin  and  formol  fuchsin.  In  sections  stained  with 
carbol  thionin  the  bacilli  are  apt  to  be  decolorized  by  the  subsequent  passage  of 
the  section  through  alcohol  and  xylol.  This  may  be  avoided  by  blotting  carefully 
after  the  thionin,  then  clearing  with  xylol  or  some  oil  and  mounting.  Nicolle's 
tannin  method  is  a  good  one. 

Mallein  is  prepared  by  sterilizing  cultures  that  have  grown  in  glycerine  bouillon 
for  about  a  month  by  means  of  heat  (ioo°C.).  The  dead  culture  is  then  filtered 
through  a  Berkefeld  filter  and  the  filtrate  constitutes  mallein.  It  is  chiefly  used  as 
a  means  of  diagnosing  the  disease  in  horses.  The  reaction  consists  in  rise  of  tempera- 
ture and  local  oedema.  The  dose  is  about  i  c.c. 

Agglutination  and  complement-fixation  tests  are  also  used  for  diagnosing  glanders. 

Bacillus  Diphtheria  (Klebs  discovered,  1883 ;  Loffler  cultivated,  1884). 
— The  diphtheria  bacillus  is  found  not  only  in  the  false  membrane  which 


FIG.  27. — Bacillus  of  diphtheria.     (Xiooo.)     (Mac  Ned.) 

is  so  characteristic  of  the  disease,  but  may  be  found  in  abundance  in 
the  more  or  less  abundant  secretions  of  nose  and  pharynx.  In  studying 
the  epidemiology  of  diphtheria,  especial  attention  must  be  given  to  the 
examination  of  nasal  discharges. 

With  diphtheria  carriers  it  is  important  to  remember  that  the  crypts  of  the  tonsils 
may  harbor  the  bacilli  and  thus  protect  them  from  the  ordinary  application  of 
antiseptic  agents.  Goldberger  found  that  the  combination  of  throat  and  nose  cul- 
tures gave  much  higher  findings  with  carriers  than  either  separately.  The  nose 
cultures  gave  more  positives  than  the  throat  ones.  These  workers  only  obtained 
about  i  %  of  positives  in  4093  cases  in  Detroit,  these  being  lower  than  figures  from 


DIPHTHERIA 


I03 


other  sources.     It  is  interesting  to  note  that  32%  of  these  people  showed  pseudo- 
diphtheria  bacilli. 

Infection  of  the  larynx  and  middle  ear  are  not  very  rare.  The  mu- 
cous membrane  of  the  vagina  or  the  conjunctiva  may  also  be  infected. 
The  B.  diphtheria  may  be  in  pure  culture  lying  entangled  in  the  fibrin 
meshes  or  contained  within  leukocytes  in  the  membrane  or  be  asso- 
ciated with  staphylococci,  pneumococci,  or  especially  streptococci. 
These  latter  complicate  unfavorably  and  cause  the  suppurative  con- 
ditions about  the  neck.  In  fatal  cases  the  diphtheria  bacillus  may  be 
found  in  the  lungs.  Ordinarily,  however,  it  remains  entirely  local  and 
does  not  get  into  the  circulation  or  viscera. 


FIG.  28. — Diphtheria  bacilli  involution  forms.     (Kolle  and  Wassermann.) 

It  produces  soluble  absorbable  poisons  which  are  designated  toxin  in  the  case 
of  the  one  responsible  for  the  acute  intoxication,  parenchymatous  degeneration  and 
death  and  toxone  for  the  poison  which  produces  oedema  at  the  site  of  inoculation  and 
postdiphtheritic  palsy.  The  injection  of  the  soluble  poisons  alone  without  the  bacilli 
produces  the  symptoms  of  the  disease. 

The  bacilli  tend  to  appear  as  slightly  curved  rods,  showing  varying 
irregularities  in  staining,  as  banding  or  beading,  and  in  particular  the 
presence  at  either  end  of  small,  deeply  staining  dots  (metachromatic 
granules). 

These  granules  may  be  seen  in  an  eighteen-hour  culture,  but  are  more  abundant 
after  thirty-six  hours.  The  granules  are  well  seen  with  Loffler's  blue,  but  better 
with  Neisser's  method.  In  culture  the  bacilli  show  swelling  at  one  or  both  ends  or 
clubbing.  In  secretions  or  in  culture  they  show  V-shapes  or  false  branching  and, 
what  is  most  characteristic,  the  parallelism— four  or  five  bacilli  lying  side  by  side  like 


104 


STUDY  AND   IDENTIFICATION   OP  BACTERIA 


palisades.  Being  a  Gram-positive  organism  while  the  majority  of  the  other  patho- 
genic bacilli  are  Gram-negative,  it  is  of  greatest  importance  to  stain  smears  by  this 
method.  It  is  not  so  strongly  tenacious  of  the  gentian  violet  as  the  cocci,  so  decolori- 
zation  should  not  be  carried  too  far. 

The  best  medium  for  growing  it  is  Loffler's  blood-serum. 

An  egg  medium,  made  of  the  whole  egg  with  glucose  bouillon  as  described  pre- 
viously, is  as  suitable  as  Loffler's  serum.  Coagulated  white  of  egg  answers  fairly 
well,  as  will  a  hard-boiled  egg — the  shell  at  one  end  being  cracked  and  the  white  cut 
with  a  sterile  knife.  This  smooth  side  is  then  inoculated  and  the  egg  placed  cut 
side  downward  in  a  sherry  glass.  If  an  incubator  is  not  at  hand  a  tube  may  be 
carried  next  the  body  in  a  pocket.  The  bacillus  grows  better  on  glycerine  agar  than 
on  plain  agar.  On  such  plates  they  appear  as  small,  coarsely  granular  colonies  with 
a  central  dark  area.  In  size  the  colonies  resemble  the  streptococcus.  On  blood- 
serum  the' colonies  are  larger — ^2  to  ^  inch  in  diameter. 


FIG.  29. — B.  diphtheria  stained  by  Neisser's  method.     (Mac  Ned.} 

The  diphtheria  bacillus  grows  luxuriantly  on  blood  agar  and  like  the  Streptococcus 
pyogenes  has  a  yellowish  laked  zone  around  the  colony.  The  Hofmann  and  the 
xerosis  bacillus  do  not  seem  to  have  this  haemolytic  power.  In  bouillon  it  tends 
to  form  a  surface  growth.  It  is  at  the  surface  that  the  toxin  function  is  most  marked, 
hence  in  growing  diphtheria  for  toxin  formation  we  use  Fernbach  flasks  which  expose 
a  large  surface  to  the  air.  It  is  a  marked  acid  producer — bouillon  with  a  -f- 1  reac- 
tion becoming  +2.5  to  +3  in  thirty-six  hours.  The  nitrate  from  a  two  or  three 
weeks'  old  broth  culture  is  highly  toxic,  and  is  usually  referred  to  as  diphtheria  toxin. 
It  is  used  in  injecting  horses  to  produce  antitoxin.  Ehrlich  uses  as  a  standard  to 
measure  the  toxicity  of  toxin  the  minimal  lethal  dose  (M.  L.  D.).  This  is  the  amount 
of  toxin  which  will  kill  a  250-gram  guinea-pig  in  just  four  days.  Some  toxins  have 
been  produced  whose  M.  L.  D.  was  ^Oo  c.c.  or  0.002  so  that  i  c.c.  of  such  toxin  would 
kill  500  guinea-pigs.  Theoretically,  the  measure  of  an  antitoxin  unit  is  the  capacity 
of  neutralizing  200  units  of  a  pure  toxin.  (On  exposure  to  light,  etc.,  toxin  loses  its 
toxic  power  and  is  termed  toxoid.) 


DIPHTHERIA  TOXIN  105 

Almost  invariably  a  bouillon  filtrate  contains  toxones  and  toxoids  besides  the  toxin. 
For  all  practical  purposes  it  is  usual  to  consider  an  antitoxin  unit  as  that  amount 
which  will  neutralize  100  M.  L.  D.  (theoretically  200  units).  Thus  it  would  be 
the  amount  which  would  neutralize  0.2  c.c.  of  the  above  noted  toxin.  In  testing 
filtrates  as  to  their  toxicity  we  make  use  of  two  limits,  one  designated  Lo  and  the 
other  L+.  When  we  add  increasing  amounts  of  a  filtrate  containing  toxins  and 
toxones  (the  toxoids  are  less  important  because  they  do  not  show  either  the  local 
reaction  or  palsy  of  toxones  and  do  not  possess  the  acute  death-producing  power  of 
the  toxin;  they  do,  however,  have  combining  power  for  antitoxin  and  are  complicating 
factors  in  standardization)  to  an  antitoxin  unit  we  gradually  reach  a  point  where  the 
slightest  further  increase  will  bring  about  slight  reaction  at  site  of  inoculation  of 
the  guinea-pig  and  possibly  some  slight  paralysis.  These  symptoms  are  due  to 
toxone  action.  The  amount  of  the  toxic  broth  filtrate  which  is  completely  neu- 
tralized by  one  antitoxin  unit  is  called  Lo.  Upon  further  addition  of  the  filtrate 
to  this  Lo  amount  we  finally  reach  a  point  when  death  of  the  25o-gram  guinea-pig 
occurs  in  four  days.  This  is  called  the  L+  or  fatal  dose.  Instead  of  having  to  add 
only  that  amount  of  filtrate  which  is  capable  of  killing  the  guinea-pig  (i  M.  L.  D.), 
it  is  found  that  we  must  add  an  amount  sufficient  to  kill  10  to  20  pigs.  The  explana- 
tion is  that  while  toxin  and  toxone  both  have  power  to  combine  with  antitoxin  and 
to  be  neutralized,  toxin  has  greater  affinity  and  can  dispossess  toxone  of  its  attach- 
ment. Consequently  when  all  the  combining  strength  of  the  antitoxin  unit  (equal 
to  100  M.  L.  D.)  has  neutralized  the  toxins  and  toxones  added  to  it  there  is  a  complete 
blocking  of  injurious  action  of  toxins  or  toxones.  When  adding  more  filtrate  to  the 
fixed  amount  of  antitoxin  a  toxin  molecule  dispossesses  a  toxone  molecule  of  its  hold 
on  the  antitoxin  unit.  The  toxin  is  neutralized  but  a  toxone  is  put  in  circulation 
and  is  capable  of  causing  reaction  and  palsy.  Not  until  every  toxone  molecule  has 
been  displaced  can  the  further  addition  of  i  M.  L.  D.,  containing  sufficient  toxin  to 
kill,  be  free  from  the  neutralizing  antitoxin  and  capable  of  producing  death  in  a 
250-gram  guinea-pig  in  four  days.  There  have  been  prepared  at  the  Hygienic 
Laboratory  in  the  U.  S.  and  in  various  European  laboratories,  by  laborious  testing 
of  antitoxic  serum,  standardized  antitoxin.  By  keeping  dried  antitoxic  serum  in  a 
vacuum  tube  under  conditions  preventing  exposure  to  heat,  light  and  moisture  the 
strength  of  the  antitoxin  unit  remains  stable.  It  may  be  stated  that  standardized 
toxin  soon  tends  to  change  in  potency,  therefore  it  is  customary  to  take  exactly 
i  unit  of  antitoxin  and  by  adding  to  it  increasing  amounts  of  toxin  to  determine  the 
amount  which  will  be  sufiicient  to  kill  the  guinea-pig  in  four  days.  This  is  usually 
somewhat  over  the  amount  which  will  kill  100  guinea-pigs,  or  100  M.  L.  D.,  and  is 
designated  the  L+  dose  of  toxin.  In  practical  application  at  biological  product 
institutions  the  L+  dose  is  tested  with  increasing  amounts  of  antitoxic  serum,  as 
drawn  from  the  immunized  horse,  and  that  amount  of  serum  which  when  mixed 
with  the  L+  dose  of  toxin  just  allows  death  of  the  guinea-pig  in  four  days  is  accepted 
as  one  antitoxin  unit. 

In  the  preparation  of  antitoxin  horses  are  employed;  the  method  being  to  inject 
the  bouillon  filtrate  or  toxin  subcutaneously  at  weekly  intervals  for  a  period  of  three 
or  four  months.  When  each  c.c.  of  the  serum  of  the  horse  is  found  to  contain  about 
250  to  500  antitoxin  units  the  horse  is  bled  from  the  jugular  vein.  Some  sera  contain 
as  much  as  1300  units  in  a  cubic  centimeter. 

Methods  of  purifying  and  concentrating  antitoxin  are  now  employed  by  certain 


106  STUDY  AND   IDENTIFICATION  OF  BACTERIA 

makers,  the  principle  being  that  the  antitoxin  in  the  horse  serum  is  precipitated 
with  the  globulins  which  come  down  on  half  saturation  with  ammonium  sulphate. 
In  this  way,  as  the  content  in  horse-serum  proteids  is  lessened,  the  anaphylactic 
dangers  are  lessened. 

As  a  curative  measure,  from  2500  to  5000  units  should  be  injected. 
If  the  injection  is  delayed  or  the  case  very  serious  the  dose  should  be 
10,000  units.  As  much  as  50,000  units  has  been  given  in  severe  cases. 
The  prophylactic  dose  is  500  units. 

Schick  Reaction. — By  the  employment  of  this  reaction  we  can  under- 
stand why  one  child  develops  clinical  diphtheria  and  another  only  shows 
the  organism  in  the  throat  (laboratory  diphtheria).  We  find  that  cer- 
tain persons  have  sufficient  amount  of  diphtheria  antitoxin  normally  in 
the  circulation  to  protect  against  the  soluble  toxin  elaborated  by  the 
organisms  localized  in  throat  or  nose.  Such  cases  show  either  a  mini- 
mal or  negative  reaction. 

Persons  not  having  any  antitoxin  in  the  circulation  show  a  positive  reaction. 
The  test  is  performed  as  follows:  With  a  small  sharp  hypodermic  needle  we  inject 
intradermally  ^0  °f  a  minimum  lethal  dose  (i  M.  L.  D.)  of  diphtheria  toxin  as 
determined  for  a  25o-gram  guinea-pig.  The  standardized  toxin  is  so  diluted  with 
a  ^%  carbolic  acid  solution  that  o.i  c.c.  contains  ^so  of  a  M.  L.  D.  A  positive 
reaction  shows  within  twenty-four  hours,  reaching  its  maximum  intensity  in  two 
days,  as  a  reddened  area,  about  i  inch  in  diameter  with  more  or  less  induration. 

The  reaction  persists  for  about  a  week,  leaving  a  brownish  pigmentation.  Positive 
reactions  show  that  the  patient  has  less  than  %Q  oi  a.  unit  of  antitoxin  in  i  c.c.  of 
his  blood-serum  and  that  he  possesses  no  immunity  to  diphtheria. 


This  test  is  of  great  value  as  showing  the  cases  needing  prophylactic 
injections  of  antitoxin.  Furthermore  nurses  showing  a  positive  reac- 
tion should  not  take  care  of  diphtheria  patients.  Carriers  of  true 
diphtheria  usually  show  a  negative  reaction  as  contrasted  with 
pseudodiphtheria  ones. 

It  is  of  value  in  showing  duration  and  degree  of  immunity  following  antitoxin 
injections  and  such  investigations  have  shown  that  intravenous  injections  are  the 
most  efficient,  next  the  intramuscular  and  least  efficient  the  subcutaneous  route. 
Moody  obtained  an  average  'of  45.2%  positives  in  524  people  examined. 

Sudden  death  after  administration  of  antitoxin  has  been  reported  in 
cases  of  status  lymphaticus.  (See  anaphylaxis.) 

Laboratory  Diagnosis. — In  obtaining  material  from  a  throat,  be  sure  that  an 
antiseptic  gargle  has  not  been  used  just  prior  to  taking  the  throat  swab.  The  part 
of  the  swab  which  touched  the  membrane  or  suspicious  spot  should  come  in  contact 
with  the  serum  slant.  This  is  best  accomplished  by  revolving  the  swab.  An  im- 


DIPHTHEROIDS  Ioy 

mediate  diagnosis  is  possible  in  probably  35%  of  cases  by  making  a  smear  from  a 
piece  of  membrane.  In  doing  this  Neisser's  stain  or  the  toluidin  blue  stain  are 
usually  considered  the  most  satisfactory.  I  prefer  the  Gram  stain,  however.  The 
diphtheria  bacilli  found  in  such  smears  are  not  apt  to  be  clubbed  and  stain  more 
uniformly. 

If  there  is  any  doubt  about  the  nature  of  an  organism  in  a  throat  culture,  always 
stain:  i.  with  Loffler's  alkaline  methylene  blue  for  two  minutes;  2.  with  Gram's 
method,  being  careful  not  to  carry  the  decolorization  too  far,  and  3.  by  Neisser's 
method.  With  Loffler's  you  obtain  a  picture  which,  after  a  little  experience,  is 
characteristic;  at  times  the  polar  bodies  show  as  intense  blue  spots  in  the  lighter 
blue  bacillus.  One  is  liable  to  confuse  cocci  lying  side  by  side  for  diphtheria  bacilli 
with  segmental  or  banded  staining.  This  difficulty  is  not  apparent  when  Gram's 
staining  is  used.  This  gives  us  great  information,  as  the  diphtheria  and  the  pseudo- 
diphtheria  are  the  only  small  Gram-positive  bacilli  usually  found  in  the  mouth. 
The  cocci  are  also  well  brought  out.  Neisser's  stain  gives  a  picture  which,  when 
satisfactory,  is  almost  absolutely  characteristic.  You  have  the  bright  blue  dots 
lying  at  either  end  of  the  light  brownish-yellow  rods.  When  first  isolated  from  a 
throat,  the  diphtheria  bacillus  is  apt  to  stain  characteristically  by  Neisser.  Later 
on,  in  subculture,  there  may  be  no  staining  of  the  polar  bodies.  Neisser  originally 
recommended  five  seconds'  application,  with  an  intermediate  washing,  for  each  of 
his  two  solutions.  Thirty  seconds  for  each  is  probably  preferable.  Some  authorities 
recommend  five  to  thirty  minutes.  It  is  well  to  bear  in  mind  that  about  2%  of  the 
people  in  apparent  health  carry  diphtheria  bacilli  of  the  granular  or  barred  type  in 
their  throats  and  of  these  about  one  in  five  will  prove  virulent  for  the  guinea-pig. 

It  is  essential  when  a  question  exists  as  to  the  nature  of  a  diphtheria- 
like  organism  to  test  it  as  to  virulence.  While  there  are  exceptions, 
especially  in  freshly  isolated  colonies,  yet  as  a  rule  a  severe  infection 
yields  virulent  organisms  and  vice  versa.  Pure  cultures  are  best  ob- 
tained by  streaking  material  from  the  throat  on  glycerine  agar  plates. 
From  an  isolated  colony  inoculate  a  tube  of  bouillon.  From  such  a  forty- 
eight-  or  seventy-two-hour-old  culture  inoculate  a  guinea-pig  with  2  or  3 
drops  subcutaneously  in  the  shaven  abdomen.  Escherich  considers  a 
fatal  result  with  1.5  c.c.  of  such  a  bouillon  culture  a  satisfactory  test  as  to 
virulence.  After  death,  which  occurs  in  two  or  three  days,  the  adrenals 
are  enlarged  and  haemorrhagic.  The  diagnosis  is  more  sure  if,  in  addi- 
tion to  the  first  animal,  a  second  one,  which  has  had  antitoxin,  is  inocu- 
lated. The  protected  one  should  live. 

Diphtheroid  Bacilli.  Pseudodiphtheria  Bacillus.  Hofmann's  Bacil- 
lus.— Under  these  terms  various  Gram-positive  bacilli  have  been  de- 
scribed as  occurring  in  genito-urinary,  nasal  and  skin  diseases. 

Their  chief  importance  is  in  connection  with  their  presence  in  the 
throats  of  healthy  people.  Probably  approximately  10%  of  people 
harbor  such  organisms  as  against  i  to  2%  with  granule  types.  Some 


108  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

authorities  believe  it  possible  for  these  diphtheroids  to  be  capable  of 
being  transformed  into  virulent  diphtheria  bacilli.  This  seems  im- 
probable. Such  organisms  are  often  found  in  urethral  discharges, 
either  alone,  or  with  gonococci  or  other  organisms. 

Recently  a  great  deal  of  attention  has  been  given  to  the  etiological  relationship 
between  diphtheroids  and  Hodgkin's  disease.  Fox,  in  a  critical  study  of  this 
relationship,  has  obtained  diphtheroids  of  varying  morphology  and  cultural  charac- 
teristics from  such  glands  as  well  as  from  enlarged  glands  of  chronic  atrophic  arthritis 
and  other  conditions.  It  would  appear  conservative  to  reject  the  acceptance  of 
diphtheroids  as  causative  agents  not  only  in  this  disease  but  in  leprosy  as  well. 

Negri  has  applied  the  name  Coryne  bacterium  granulatis  malignl  to  diphtheroids 
isolated  from  glands  in  Hodgkin's  disease.  The  granular  rods  of  Much,  supposed 
to  be  connected  with  tubercle  bacilli,  may  be  diphtheroids.  Mallory  has  connected 
diphtheroids  with  scarlet  fever. 

DIPHTHEROID  CHARACTERISTICS 

1.  They  very  rarely  give  the  blue  dot  staining  at  the  two  ends.     Exceptionally 
they  may  give  a  dot  at  one  end.     Neisser  attaches  importance  to  the  dots  at  both 
ends  as  showing  diphtheria. 

2.  They  tend  to  stain  solidly  or  at  most  with  only  a  single  unstained  segment. 
They  are  shorter,  thicker,  and  do  not  curve  so  gracefully  as  the  true  diphtheria 
bacillus.    They  are  stockier. 

3.  They  produce  very  little  acid  in  sugar  media,  not  one-half  that  produced  by 
true  diphtheria.     Goldberger  found  29  out  of  30  cultures  of  B.  diphtheria  virulent 
and  acid  producers.     Of  47  Hofmann  cultures  6  showed  slight  acid  production  while 
41  produced  alkali.     All  were  nonvirulent. 

4.  They  are  nonpathogenic  for  guinea-pigs. 

5.  Many  of  them  grow  quite  luxuriantly  and  often  show  chromogenic  power. 

Xerosis  Bacillus. — This  organism  is  frequently  found  in  normal  con- 
junctival  discharges.  There  is  question  as  to  its  pathogenesis,  and  the 
finding  of  this  organism  should  not  exclude  the  previous  presence  of 
strictly  pathogenic  organisms,  such  as  the  Gonococcus  or  the  Koch- 
Weeks.  It  resembles  the  diphtheria  bacillus  in  being  Gram-positive 
and  showing  parallelism,  but  differs  i.  in  being  nonvirulent  for  guinea- 
pigs;  2.  in  requiring  about  two  days  for  the  appearance  of  colonies;  3. 
in  not  showing  Neisser's  granule  staining,  and  4.  in  producing  very 
little  acid  in  sugar  media. 


CHAPTER  VIII 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.    GRAM- 
NEGATIVE  BACILLI.    KEY  AND  NOTES 

KEY  to  the  recognition  of  nonspore-bearing,  nonchromogenic,  non- 
Gram-staining,  nonbranching  bacilli. 

(NOTE. — Some  books  say  that  the  proteus  group  is  Gram-positive.     It  is,  how- 
ever, usually  negative.) 
Do  not  grow  on  ordinary  media.     Require  blood  agar  (haemophilia  bacteria),  serum 

agar,  or  blood-serum. 

Minute  dewdrop  colonies. 

1.  Influenza  bacillus.     Requires  blood  media. 

2.  Koch- Weeks  bacillus  (conjunctivitis).     Serum  agar  best  medium.     Many, 
however,  regard  haemoglobin  as  necessary  for  growth. 

3.  Muller's  bacillus  of  trachoma.    Like  Koch- Weeks  bacillus,  but  easier  to 
^cultivate. 

4.  Morax  diplobacillus  of  conjunctivitis.     Grows  well  and  produces  little  pits 
of  liquefaction  in  Loffler's  blood-serum. 

5.  Bordet-Gengou  bacillus  of  whooping-cough.     Does  not  grow  on  LofBer's 
serum.     Requires  blood  or  ascitic  fluid  agar.     Original  isolation  should  be  on 
glycerine  potato  agar. 

6.  Ducrey's  bacillus  (soft  chancre).     Requires  media  rich  in  blood  or  serum. 
Forms  chains. 

Grow  well  on  ordinary  media. 

I.  Cultures  in  litmus  milk.     PINK. 

A.  Nonmotile. 

Lactis  aerogenes  group.     B.  lactis  aerogenes. 

Produce  gas  in  glucose,  lactose,  and  saccharose.  No  liquefaction  of  gelatin. 
Short,  stubby  bacteria,  often  showing  capsules.  Intermediate  between  the 
colon  and  Friedlander  group. 

B.  Motile. 

1.  Nonliquef action  of  gelatin. 

(a)  B.  coli  group.  Coagulation  of  milk.  No  subsequent  peptonization. 
Gas  in  glucose  and  lactose,  none  in  saccharose.  Indol  produced. 
Neutral  red  reduced. 

2.  Liquefaction  of  gelatin. 

(a)  B.  cloaca?  group.     Gas  in  glucose,  slight  in  lactose.     Slow  coagulation 
of  milk.     Subsequent  peptonization. 
109 


110  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

II.  Cultures  in  litmus  milk.    LILAC. 
A.  Nonmotile  bacilli. 

1.  No  gas  generated  in  glucose  or  lactose  bouillon. 

(a)  Haemorrhagic  septicaemia  group.     These  are  oval  bacilli  with  tend- 
ency to  bipolar  staining. 

Colonies  smaller  and  less  opaque  than  those  of  B.  coli. 

Examples:  B.  pestis,  B.  suisepticus,  B.  cholerae  gallinarum  (chicken 

cholera). 

B.  pseudotuberculosis  rodentium  (very  similar  to  plague). 

B.  pestis  is  absolutely  nonmotile,  does  not  liquefy  gelatin,  does  not 

produce  indol,  produces  slight  acid  in  glucose  but  not  in  lactose 

bouillon. 

(b)  Dysentery  group.     Colonies  similar  to  those  of  B.  coli. 
Divided  into  two  classes  according  as  mannite  is  acted  on : 
Those  not  giving  acid — nonacid  group — (Shiga-Kruse). 
Those  giving  acid — acid  group — (Flexner-Strong). 

2.  Gas  generated  in  glucose  bouillon  not  in  lactose. 

(a)  Friedlander  group.     Give  very  viscid,  porcelain-like  colonies. 
Tendency  to  capsule  formation  in  favorable  media. 
Examples:  B.  pneumonias,  B.  capsulatus  mucosus,  B.  rhinoscleroma 
B.  Motile  bacilli. 

1.  Do  not  liquefy  gelatin. 

(a)  Do  not  produce  gas  in  either  glucose  or  lactose  bouillon. 
Typhoid,  or  Eberth  group.     No  indol.     No  coagulation  of  milk.     No 
reduction  of  neutral  red. 

(b)  Gas  generated  in  glucose,  not  in  lactose  media.     Milk  not  coagulated. 
Neutral  red  reduced. 

Gartner  group.     This  includes: 

Pathogenic  types  for  man;  as  B.  enteritidis,  B.  icteroides,  B.  para- 
typhoid B,  B.  psittacosis.  Nonpathogenic  for  man;  as  B.  cholerae 
suum  (hog  cholera). 

2.  Liquefy  gelatin. 

(a)  Proteus  group.     Colonies  at  first  round  later  amoeboid,  spreading. 
Produce  gas  in  glucose,  not  in  lactose.     Produces  foul  odor. 
B.  zopfii  type  of  Proteus  group  does  not  liquefy  gelatin;  colonies  at  first 
round,  later  amoeboid,  spreading.     Foul  odor  in  cultures.     Gelatin 
stab  shows  lateral  branching. 

NOTE. — The  Friedlander  and  the  lactis  aerogenes  group,  differ  culturally  chiefly 
in  carbohydrate  fermentation  activities  and  organisms  considered  as  belonging 
to  the  Friedlander  group  rather  than  to  the  lactis  aerogenes  group  may  show  acid 
in  litmus  milk.  Where  an  organism  having  the  characteristics  of  B.  coli,  but  fer- 
menting saccharose,  is  found,  it  is  termed  B.  coli  communior.  A  nongas  producing 
colon  type  organism  has  been  designated  B.  coli  anaerogenes.  Certain  organisms 
which  turn  litmus  milk  lilac  and  which  liquefy  gelatin,  but  do  not  produce  gas  in 
sugar  media,  belong  to  the  "Booker"  group.  Other  organisms  which  acidify  and 
coagulate  litmus  milk  but  do  not  liquefy  gelatin  or  produce  gas  in  glucose  or  lactose 
media  have  been  placed  in  the  "Bienstock"  group.  The  proteus  or  Hauser  group  is 


INFLUENZA  III 

composed  of  organisms  showing  various  functions;  Prcteus  vulgaris  liquefying  gelatin 
rapidly,  P.  mirabilis  slowly  and  P.  zenkeri  not  at  all.  The  differentiation  of  the  colon 
group  is  extensively  considered  under  bacteriology  of  water. 

GRAM-NEGATIVE  BACILLI  REQUIRING  SPECIAL  MEDIA 

Bacillus  Influenzas  (Pfeiffer,  1892). — This  organism  is  the  type  of  the 
so-called  haemophilia  bacteria — organisms  whose  growth  is  restricted 
to  media  containing  haemoglobin.  The  influenza  bacillus  seems  to  grow 
better  on  slants  freshly  streaked  with  blood  than  on  those  which  have 
been  made  for  some  time,  and  they  appear  to  grow  better  on  this  sur- 
face smear  of  blood  than  on  a  mixture  of  agar  and  blood. 

The  influenza  bacilli  are  most  likely  to  be  isolated  from  the  sputum  of  broncho- 
pneumonia  due  to  this  organism.  It  has  also  frequently  been  found  in  the  nasal 
secretions  of  influenza  patients.  Exceptionally,  it  is  present  in  the  blood,  and  has 
been  isolated  in  cases  of  meningitis  from  cerebrospinal  fluid.  It  also  occurs  at  times 
in  anginas,  but  then  usually  associated  with  other  organisms.  Infection  probably 
only  takes  place  by  contact.  It  is  a  very  small  bacillus  which  in  sputum  tends  to 
show  itself  in  aggregations,  especially  centering  about  M.  tettragenus.  It  stains  rather 
faintly  when  compared  with  cocci,  so  that  a  smear  of  sputum  stained  with  formol 
fuchsin  shows  a  deep  violet  staining  for  the  M.  tetragenus  or  other  cocci,  and  scattered 
around  in  a  clump-like  aggregation  we  see  these  minute,  rather  faintly  stained  rods. 
They  also  tend  to  stain  more  deeply  at  either  end,  so  that  they  sometimes  appear 
as  diplococci.  Gram's  method,  counterstaining  with  formol  fuchsin  is  excellent  for 
their  demonstration.  The  red  bacilli  and  the  violet-black  cocci  are  easily  dis- 
tinguished . 

To  cultivate  them,  rub  the  sputum,  or  at  autopsy  the  material  from 
a  lung,  on  a  slant  smeared  with  human  blood  (pigeon's  blood  is  also 
satisfactory),  and  then  without  sterilizing  the  loop,  inoculate  a  second 
blood  slant;  then  a  third,  and  possibly  a  fourth. 

The  colonies  appear  as  very  minute  dewdrop-like  points  which  seem  to  run^into 
each  other  in  a  wave-like  way.  To  test  such  colonies  we  should  transfer  a  single 
colony  to  plain  agar  and  blood-serum,  trying  not  to  carry  over  any  blood.  If  the 
least  trace  of  blood  is  carried  over,  they  may  grow  on  agar  or  blood-serum.  Organ- 
isms resembling  the  influenza  bacillus  have  been  isolated  from  whooping-cough. 
Such  organisms  have  also  been  found  in  the  fauces  of  well  persons.  In  many 
epidemics  of  influenza  the  bacillus  has  not  been  isolated,  or  success  has  obtained  in 
only  a  small  proportion  of  the  cases.  Etiological  factors  in  conditions  more  or  less 
resembling  influenza  may  be  the  Streptococcus,  Pneumococcus,  or  M.  calarrhahs. 
The  influenza  bacillus  seems  to  grow  best  in  symbiosis  with  some  other  organism, 
especially  with  S.  pyogenes  aureus. 

Koch-Weeks  Bacillus  (Koch,  1883).— This  produces  a  severe  con- 
junctivitis. It  is  very  common  in  Egypt  and  is  also  a  frequent  cause 
of  conjunctivitis  in  the  Philippines  and  in  temperate  climates. 


112  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

Smears  made  from  conjunctival  secretion  show  large  numbers  of  small  Gram- 
negative  bacilli,  especially  contained  within  pus  cells,  but  also  lying  free.  They  are 
more  difficult  to  cultivate  than  the  influenza  bacillus,  but  the  same  general  methods 
hold.  The  vitality  of  this  organism  is  very  slight  so  that  almost  immediate  trans- 
ference of  material  is  necessary.  Flies  are  an  important  factor  in  Egypt.  The 
period  of  incubation  is  short,  twelve  to  thirty-six  hours.  The  best  medium  is  a 
mixture  of  glycerine  agar  and  hydrocele  or  ascites  fluid.  At  first  we  rarely  obtain 
pure  cultures.  The  colonies  are  dewdrop-like  and  first  show  themselves  in  about 
thirty-six  hours  in  incubator  cultures. 

It  would  seem  that  blood  agar  is  a  better  medium  than  a  serum  one. 
Many  haemoglobinophilic  bacteria  will  grow  with  only  i  to  500  haemo- 
globin, so  that  growth  on  serum  might  be  explained  by  slight  blood 
admixture. 

Some  have  thought  that  repeated  infection  with  the  Koch- Week's  bacillus  was 
the  cause  of  trachoma.  Others  have  regarded  other  haemoglobinophilic  bacteria  as 


FIG.  30.— The  Koch- Weeks  Bacillus.     (Hansell  and  Sweet.) 

causative.  According  to  the  views  of  Park  and  Williams  the  inclusion  bodies  of 
Prowazek,  supposed  to  be  characteristic  of  trachoma,  are  simply  clumps  of  extremely 
small,  coccoid  haemoglobinophilic  bacteria.  Besides  these  organisms  the  Gonococcus 
in  smears  from  gonorrhceal  ophthalmia  is  stated  to  show  involution  forms,  making 
a  resemblance  to  trachoma  bodies. 

Noguchi  thinks  that  while  there  is  a  morphological  similarity  between  degenerated 
haemoglobinophilic  bacteria  and  cell  inclusion^,  yet  in  the  latter,  the  elementary 
bodies  are  much  smaller  than  the  bacterial  granules,  and  the  initial  bodies  less  definite 
in  contour.  He  was  able  to  infect  the  conjunctivae  of  monkeys  with  inclusion  bodies 
material,  but  not  with  haemoglobinophilic  bacilli. 

Diplobacillus  of  Morax. — This  organism  causes  mild  blepharo-con- 
junctivitis  chiefly  at  the  internal  angle  of  the  eye.  They  are  about  i  or 


PERTUSSIS 

2/z  long  and  tend  to  occur  in  pairs  or  short  chains.     Some  claim  that 
they  are  Gram-positive. 

Culturally  the  formation  of  little  pits  of  liquefaction  in  Loffler's  serum  within 
twenty-four  hours  which  later  become  confluent  may  be  regarded  as  fairly  character- 
istic. They  do  not  grow  on  nutrient  agar. 

After  two  or  three  days  on  blood-serum  rather  marked  involution 
forms  occur.  While  usually  causing  a  more  or  less  chronic  conjuncti- 
vitis they  may  at  times  produce  a  keratitis. 

NOTE. — A  Gram-negative  bacillus  which  is  less  than  i  micron  long,  growing  singly, 
or  in  pairs,  and  known  as  the  bacillus  of  Zur  Nedden  has  been  stated  to  produce 
corneal  ulcers.  It  grows  readily  on  agar  or  other  ordinary  culture  media.  It 
coagulates  milk. 

Bacillus  of  Chancroid  (Ducrey,  1889). — These  are  short  coccobacilli, 
occurring  chiefly  in  chains.  They  show  bipolar  staining.  They  grow 
best  in  a  mixture  of  blood  and  bouillon. 

Material  for  culturing  should  be  obtained  before  the  lesion  ulcerates.  The 
exudate  should  be  inoculated  on  blood  agar,  i  part  blood  to  2  of  agar.  After  forty- 
eight  hours  small  glistening  colonies  develop  which  easily  slide  about  the  slant  when 
touched  with  a  loop. 

Bacillus  of  Bordet-Gengou. — This  bacillus  was  reported  as  the  cause 
of  whooping-cough  by  Bordet  and  Gengou  in  1906.  (Czaplewski  and 
Reyher  had  previously  reported  oval  bipolar  staining  organisms,  as  the 
cause  of  pertussis,  and  other  authors  influenza-like  organisms.) 

The  bacillus  is  oval,  Gram-negative,  shows  bipolar  staining,  somewhat  resembles 
B.  influenza  and  grows  only  on  uncoagulated  serum  media,  as  blood  or  ascites  agar. 
The  original  cultures  are  very  scanty  so  that  the  colonies  are  difficult  to  recognize. 
In  subcultures  the  growth  is  more  flourishing.  The  organism  is  only  found  in  white, 
thick,  leukocyte  abounding  sputum,  of  the  beginning  of  the  disease.  Hence  per- 
tussis is  probably  contagious  only  at  the  onset. 

Complement  binding  and  agglutination  reactions  have  been  obtained.  For 
diagnosis  stain  the  sputum.  Remember  that  pertussis  gives  a  mononuclear  leuko- 
cytosis  of  15,000  to  50,000. 

For  isolation  from  sputum  the  following  medium  is  required.  Auto- 
clave 500  grams  potato  with  1000  c.c.  of  4%  glycerine  solution.  Pour 
off  excess  of  fluid.  Emulsify  potato  in  1 500  c.c.  normal  salt  solution  and 
add  powdered  agar  to  3  or  4%.  For  use  mix  with  an  equal  quantity 
of  defibrinated  blood. 


114  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

GRAM  NEGATIVE  BACILLI  GROWING  ON  ORDINARY  MEDIA 

Bacillus  Pneumonias  (Friedlander,  1882). — This  organism  is  re- 
sponsible for  about  5%  of  the  cases  of  pneumonia.  It  is  usually 
termed  the  Pneumobacillus  to  distinguish  it  from  the  Pneumococcus; 
at  other  times  Friedlander's  bacillus.  The  name  of  Fraenkel  attaches 
to  the  Pneumococcus.  Morphologically,  it  is  a  short,  thick  bacillus, 
and  in  pathological  material,  as  sputum,  shows  a  wide  capsule.  It  is 
nonmotile  and  Gram-negative.  The  colonies  on  agar  are  of  a  pearly 
whiteness  and  are  markedly  viscid.  On  potato  it  shows  a  thick  viscid 
growth  containing  gas  bubbles.  The  characteristic  culture  is  the  nail 
culture  of  a  gelatin  stab.  The  growth  at  the  surface  is  heaped  up  like  a 
round-headed  nail,  the  line  of  puncture  resembling  the  shaft  of  the  nail. 

It  does  not  liquefy  gelatin.  It  does  not  produce  indol,  and  does  not  produce  gas 
in  lactose  bouillon — differences  from  the  colon  bacillus — with  which  it  may  be  con- 
fused in  cultures,  as  it  does  not  then  possess  a  capsule.  If  in  doubt,  inject  a  mouse 
at  the  root  of  the  tail.  Death  from  septicaemia  occurs  in  two  days.  The  peritoneum 
is  sticky  and  numerous  capsulated  bacilli  are  present  in  the  blood  and  organs.  The 
organisms  which  have  been  isolated  from  rhinoscleroma  and  ozcena  are  practically 
identical  with  the  B.  pneumonia.  This  group  of  organisms  is  generally  referred 
to  as  the  Friedlander  group.  Similar  organisms  have  been  isolated  from  the  dis- 
charges of  middle-ear  diseases  and  in  anginas.  Cases  have  been  reported  where  the 
B.  pneumonia  was  the  cause  of  septicaemia  in  man. 

Bacillus  Pestis  (Kitasato,  Yersin,  1894).— This  is  the  organism  of 
plague.  It  is  primarily  a  disease  of  rats.  It  is  the  member  of  the  group 
of  haemorrhagic  septicaemias  (Pasteurelloses),  from  which  man  suffers. 

Other  Pasteurelloses  are  chicken  cholera,  swine  plague,  mouse  sep- 
ticaemia and  rabbit  septicaemia.  This  is  a  widely  distributed  group  and 
may  include  saprophytic  organisms  as  well  as  those  noted  for  their 
virulence. 

B.  cholera  gallinarum  and  B.  suisepticus  are  approximately  similar  in 
size  and  cultural  requirements  to  B.  pestis.  The  oval  bacillus  with 
bipolar  staining  in  smears  from  tissues  is  very  characteristic  for  both 
of  them.  Another  name  for  swine  plague  (B.  suisepticus)  is  infectious 
pneumonia  of  swine.  The  organism  is  chiefly  found  in  the  lungs.  The 
bacillus  of  plague  was  first  isolated  by  Yersin  from  a  plague  bubo,  in 
1894,  at  Hong  Kong.  It  is  true  that  Kitasato  reported  a  bacillus 
which  he  had  isolated  from  the  blood  of  a  plague  patient,  on  July  7, 
1894  (Yersin's  report  was  made  July  30,  1894).  Kitasato's  bacillus 
was  motile,  Gram-positive,  coagulated  milk  and  gave  a  turbidity  in 


PLAGUE  115 

bouillon,  characteristics  which  were  just  the  opposite  of  those  of  the 
organism  reported  by  Yersin. 

Where  the  plague  bacilli  are  found  chiefly  in  the  glands,  we  have  bubonic  plague; 
when  in  lungs,  pneumonic  plague;  when  localized  in  the  skin  and  subcutaneous  tissue, 
the  cellulo-cutaneous;  and  when  as  a  septicaemia,  septicaemic  plague.  An  intestinal 
type  is  recognized  by  some  authors.  It  must  be  remembered  that  in  all  forms  of 
plague  the  lymphatic  glands  show  hemorrhagic  oedema;  it  is  in  bubonic  plague,  how- 
ever, that  the  areas  of  necrosis  with  periglandular  oedema  are  prominent. 

Where  the  symptoms  are  slight,  mainly  buboes,  the  term  pestis  minor 
is  sometimes  used;  the  typical  disease  being  termed  pestis  major.  In 
pneumonic  plague  we  have  a  bronchopneumonia. 

In  smears  from  material  from  buboes,  from  sputum,  or  in  blood  smears,  as  well 
as  from  blood  or  spleen  smears  from  experimental  animals,  we  obtain  the  typical 


FIG.  3 1  .—Colonies  of  plague  bacilli  forty-eight  hours  old.     (Kolle  and  Wassermann.') 

morphology  of  a  coccobacUlus  (1.5X0.5*1)  with  very  characteristic  bipolar  staining; 
there  being  an  intermediate,  unstained  area.  Very  characteristic  also  is  the  appear- 
ance in  these  smears  of  degenerate  types  which  stain  feebly  and  show  coccoid  and 
inflated  oval  forms.  The  presence  of  these  involution  forms  associated  with  typical 
bacilli  is  almost  diagnostic  for  one  with  experience.  Inoculating  tubes  of  plain  agar 
and  3%  salt  agar  with  this  same  material,  we  obtain  in  plain  agar  cultures  organisms 
which  are  typically  small,  fairly  slender  rods,  which  do  not  stain  characteristically 
at  each  end  and  are  not  oval.  The  smear  obtained  from  the  salt  agar  presents  most 
remarkable  involution  forms— coccoid,  root-shaped,  sausage-shaped  forms,  ranging 
from  3  to  12  microns  in  length,  more  resembling  cultures  of  moulds  than 
bacteria.  Another  point  is  that  on  the  inoculated  plain  agar  we  are  in  doubt  at 
the  end  of  twenty-four  hours  whether  the  dewdrop-like  colonies  are  really  bacterial 
colonies  or  only  condensation  particles.  By  the  second  day,  however,  these  colo 
have  an  opaque  grayish  appearance,  so  that  now,  instead  of  questioning  the  presi 


n6 


ST"UDY  AND   IDENTIFICATION   OF  BACTERIA 


of  a  culture,  we  consider  the  possibility  of  contamination.  Litmus  milk  is  rendered 
slightly  acid  but  not  sufficiently  to  change  the  lilac  color.  Glucose  broth  is  made 
slightly  acid  but  there  is  no  effect  on  lactose. 

Blood  cultures  in  septicsemic  plague  may  show  from  5  to  500,000  bacilli  per  c.c. 
Smears  from  the  blood  in  such  cases  are  positive  in  only  about  17%. 

The  plague  bacillus  grows  well  at  room  temperature — its  optimum 
temperature  being  30°  instead  of  37°C.,  as  is  usual  with  pathogens. 
Next  to  the  salt  agar  culture,  the  most  characteristic  one  is  the  stalac- 
tite growth  in  bouillon  containing  oil  drops  on  its  surface.  The  culture 
grows  downward  from  the  undersurface  of  the  oil  drops  as  a  powdery 


FIG.  32. — Pest  bacilli  from  spleen  of  a  rat.     (Kolle  and  Wassermann.) 

thread.     These  are  very  fragile,  and  as  the  slightest  jar  breaks  them, 
it  is  difficult  to  obtain  this  cultural  characteristic. 

While  Klein  states  that  B.  coli,  Proteus  vulgaris  and,  in  particular,  B.  bristolensis 
may  be  mistaken  for  plague  bacilli,  if  bipolar  staining  alone  be  relied  upon,  yet  it  is 
B.  pseudotuberculosis  rodentium  which  may  confuse  an  experienced  worker.  While 
this  latter  is  only  moderately  pathogenic  for  rats  yet  the  fact  that  rats  may  be 
immunized  to  B.  pestis  by  inoculation  with  B.  pseudotuberculosis  rodentium  brings 
up  the  suspicion  of  identity  of  the  two  organisms.  In  diagnosing  ^always  use  animal 
experimentation.  Owing  to  the  difficulty  in  emulsifying  plague  bacilli,  agglutination 
tests  are  not  satisfactory.  B.  tularense  resembles  B.  pestis.  See  Part  IV. 

Albrecht  and  Ghon  have  shown  that  by  smearing  material  upon  the 
intact,  shaven  skin  of  a  guinea-pig,  infection  occurs.  This  is  the  crucial 
test. 

A  pocket  made  by  cutting  the  skin  of  a  guinea-pig  with  scissors  and  extended 
subcutaneously  with  scissors  or  forceps,  into  which  a  piece  of  the  suspected  plague 


RATS   AND   PLAGUE  H^ 

tissue  is  thrust  with  forceps,  is  more  practical  than  injecting  an  emulsion  with  hypo- 
dermic syringe. 

Mice  inoculated  at  the  root  of  the  tail  quickly  succumb.  Rats,  this  being  pri- 
marily a  disease  of  rats,  are  of  course  susceptible.  Other  rodents,  as  squirrels,  are 
susceptible.  It  has  been  suggested  that  a  rodent,  the  Siberian  marmot,  or  tarabagan 
(Arctomys  bobac}  might  be  the  starting-point  of  plague  outbreaks.  In  natural 
plague  of  rats,  the  lesions  which  establish  a  diagnosis  even  without  the  aid  of  a 
microscope  are  dark  red,  subcutaneous  injection  of  the  flaps  of  the  abdominal  walls 


FIG.  33. — Pest  bacillus  involution  forms  produced  by  growing  on  3%  salt  agar. 
(Kolle  and  Wassermann.) 

as  they  are  turned  back,  fluid  in  the  pleural  cavities,  oedematous  haemorrhagic 
periglandular  infiltration  and  swelling  of  the  neck  glands,  and  in  particular  a  creamy, 
mottled  appearance  of  the  liver. 

The  bacillus  known  as  Danysz  virus  also  causes  whitish  granules  of 
liver  but  these  are  larger  and  do  not  have  the  appearance  as  if  peppered 
on  the  liver. 

The  neck  glands  in  rat  plague  are  chiefly  involved  because  the  flea  prefers  to 
inhabit  the  skin  of  the  neck.  The  spleen  is  swollen,  congested  and  granular  and 
smears  from  this  viscus  will  show  the  bacilli. 

A  chronic  rat  plague,  which  may  be  a  factor  in  keeping  up  the  disease,  is  char- 
acterized by  enlargement  of  the  spleen  and  the  presence  within  it  of  nodules  contain- 
ing plague  bacilli.  McCoy  has  noted  that  the  frequency  of  the  cervical  bubo  in 
rats,  noted  by  the  Indian  Commission  (72%),  was  not  found  in  California.  The 
glands  show  periglandular  infiltration  and  injection  as  well  as  enlargement. 

Recent  investigations  in  India  have  definitely  determined  the  fact 
that  the  flea  (Xenopsylla  cheopis)  is  the  intermediary  in  the  transmis- 
sion of  plague  from  rat  to  rat  and  from  rat  to  man. 


Il8  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

In  Europe  and  U.  S.,  Ceratophyttus  fasciatus  is  the  common  rat  flea  and  it,  as  well 
as  other  species  of  flea,  may  transmit  the  disease.  The  bedbug  will  also  transmit 
plague.  Fleas  suck  up  the  septicaemic  blood  of  infected  rats  and  there  is  a  develop- 
ment of  plague  bacilli  in  the  cesophagus  with  more  or  less  obstruction.  When 
feeding  on  man  or  other  rats  such  fleas  regurgitate  and  thus  inoculate  these  plague 
bacilli.  The  faeces  of  such  fleas  are  also  infectious. 

In  primary  pneumonic  plague  the  infective  nature  is  very  great  and 
appears  to  be  by  the  respiratory  atrium  (from  man  to  man).  This 
was  the  terrifying  type  of  plague  in  the  black  death  of  the  four- 
teenth century. 

Strong  and  Teague  have  shown  that  of  39  plates  exposed  before  the  mouths  of 
patients  with  pneumonic  plague,  with  marked  dyspnoea  and  pulmonary  oedema,  but 
without  coughing,  only  i  plate  showed  plague  bacilli.  In  39  other  experimental 
plate  cultures  with  coughing  on  the  part  of  the  patients  there  were  15  plates  showing 
plague  bacilli. 

The  droplet  method  of  infection  is  therefore  the  important  one  in  plague  pneu- 
monia. 

As  these  droplets  are  expelled  to  a  considerable  distance  not  only  should  the 
respiratory  inlets  be  protected  by  masks  but  the  conjunctivas  with  glasses  and  abra- 
sions with  protective  coatings. 

For  diagnosis  make  smears  and  cultures  from  material  drawn  from 
a  bubo  by  a  syringe.  (At  a  later  stage,  when  softening  begins,  there 
may  not  be  any  bacilli  present.)  Also,  if  pneumonic  plague,  from  the 
sputum.  Blood  cultures  and  even  blood  smears  may  be  employed  in 
septicaemic  plague.  Formol  fuchsin  and  Archibald's  stain  make  satis- 
factory stains.  Always  inoculate  a  guinea-pig  with  the  material  either 
by  rubbing  it  in  with  a  glass  spatula  on  the  shaven  skin  or  by  sub- 
cutaneous injection. 

For  prophylaxis  the  most  important  method  is  that  of  Haffkine.  Stalactite  bouil- 
lon cultures  of  plague  are  grown  for  five  to  six  weeks.  These  are  killed  by  a  tem- 
perature of  65°C.  for  one  hour.  Lysol  (M%)  is  added  to  the  preparation  and  from 
0.5  to  4  c.c.  injected,  according  to  the  age  and  size  of  the  individual  treated.  Sus- 
ceptibility is  reduced  about  one-fourth,  and  of  those  attacked  after  previous  vaccina- 
tion, the  mortality  is  only  about  one-fourth  of  what  it  is  among  the  noninoculated. 
Strong  prepares  a  prophylactic  vaccine  from  living  plague  cultures  rendered  avirulent. 
Yersin's  serum,  made  by  injecting  horses  with  dead  plague  cultures  and  afterward 
with  living  ones,  is  of  value  prophylactically  and  has  possibly  considerable  curative 
power. 

The  Eberth,  Gartner  and  Escherich  Groups. — From  a  standpoint 
of  cultures  in  litmus  milk  and  sugar  bouillon  we  can  divide  the  organ- 
isms related  to  typhoid  at  one  extreme  and  the  colon  at  the  other  into 
three  groups. 


THE  TYPHOID-COLON  GROUP 


119 


1.  The  Eberth  or  typhoid  group.     There  are  three  important  patho- 
gens in  this  group:  the  B.  typhosus,  the  B.  dysenteric,  and  the  B.  facalis 
alkaligenes.    The  color  of  litmus  milk  is  practically  unaltered  and  there 
is  no  gas  production  in  either  glucose  or  lactose  bouillon.    No  coagu- 
lation of  milk.     No  reduction  of  neutral  red.     The  B.  typhosus  and 
the  B.  facalis  alkaligenes  are  actively  motile,  while  the  B.  dysenteries 
is  nonmotile  or  practically  so. 

During  the  first  twenty-four  to  forty-eight  hours  there  is  a  moderate  acid  produc- 
tion by  typhoid,  so  that  the  milk  culture  is  less  blue,  while  with  the  B.  facalis  alka_ 
ligenes  the  alkalinity  is  intensified  from  the  start,  so  that  the  blue  color  is  deepened^ 

2.  The  Gartner  or  hog  cholera  group.     Besides  organisms  important 
for  animals  and  probably  at  times  for  man,  such  as  B.  cholera  suum 
and  B.  psittacosis  and  B.  icteroides  (interesting  historically  as  having 
been  reported  as  the  cause  of  yellow  fever  by  Sanarelli),  we  have  two 
pathogens:  i.  B.  enteritidis  (Gartner's  bacillus)  and  2.  B.  paratyphoid 
B.     In  this  connection  it  may  be  stated  that  the  present  view  is  that 
hog  cholera  is  caused  by  an  ultra-microscopic  organism  and  not  by  the 
B.  cholera  suum. 

These  organisms  cannot  be  separated  culturally,  but  only  by  immunity  reactions. 
They  do  not  turn  litmus  milk  pink.  They  produce  gas  in  glucose  bouillon,  but  not 
in  lactose.  They  very  powerfully  reduce  neutral  red  with  the  production  of  a  yellow- 
ish fluorescence.  They  do  not  coagulate  milk.  There  is  a  transient  acidity  in  the 
litmus  milk,  but  becoming  shortly  afterward  alkaline,  the  lilac-blue  color  is  intensified. 
Both  organisms  are  motile. 

3.  The  Escherich  or  colon  group.    These  turn  litmus  milk  pink, 
coagulate  milk,  reduce  neutral  red,  and  show  varying  degrees  of  motil- 
ity.     The  three  groups  of  organisms  just  described  are  nonliquefiers  of 
gelatin.     Two  intestinal  organisms,  the  B.  cloaca  and  the  Proteus  vul- 
garis,  differ  in  liquefying  gelatin. 

Bacillus  Typhosus  (Eberth,  1880;  Gaffky,  1884). — This  organism 
may  be  isolated  from  the  stools,  urine,  and  the  blood  of  typhoid  patients. 

At  postmortem  it  can  be  best  isolated  from  the  spleen,  but  is  also  present  in 
Peyer's  patches  which  have  not  ulcerated.  When  ulceration  has  occurred  contami- 
nation with  B.  coli,  is  almost  sure.  Cultures  may  be  obtained  from  the  liver  also. 
In  sections  made  from  spleen  the  Gram-negative  bacilli  are  apt  to  be  decolorized. 
Thionin,  then  blotting  and  clearing  in  oil  or  xylol,  shows  the  clumps  of  bacilli  lying 
between  the  cells. 

Formerly  it  was  supposed  that  by  the  differences  in  the  thickness  of  the  film  of 
a  colony  or  by  its  varying  shades  of  grayish-blue,  we  possessed  data  of  importance  in 
differentiating  typhoid  from  related  organisms. 


120  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

The  colonies  look  like  grapevine  leaves. 

Growth  on  potato  was  also  considered  as  affording  information.  At  present, 
the  biochemical  reactions  give  us  information  assisting  in  differentiation,  and  the 
agglutination  and  bacteriolytic  phenomena,  the  final  diagnosis.  The  various  plating 
media  are  considered  under  media  for  plating  out  faeces. 

Not  only  do  we  find  hyperplasia  of  the  endothelial  cells  in  the  lymphoid  tissue 
of  Peyer's  patches  and  the  mesenteric  glands  and  the  spleen,  with  subsequent  necro- 
ses, but  focal  necroses  of  the  same  character  are  found  in  the  liver. 

A  striking  feature  of  the  pathology  of  typhoid  fever  is  the  long-con- 
tinued persistence  of  the  organisms  in  the  gall-bladder  and  elsewhere. 
It  is  beginning  to  be  believed  that  a  previous  typhoid  infection,  pos- 
sibly so  mild  as  to  have  passed  unnoticed,  is  at  the  basis  of  gall-bladder 
infections  and  resulting  gall-stones.  Various  bone  infections,  especially 


FIG.  34. — Seventy-two-hour-old  culture  of  typhoid  bacillus  on  gelatin.     (Kolle  and 

Wassermann.) 

osteomyelitis,  have  shown  the  typhoid  bacilli  in  pure  culture.  For- 
merly it  was  supposed  that  the  typhoid  bacillus  brought  about  its  lesions 
by  a  local  infection  centered  in  the  ileum.  The  present  view  is  that 
typhoid  bacilli  effect  an  entrance  into  the  blood  stream  through  some 
lymphoid  channel,  as  by  tonsil  or  other  alimentary  lymphoid  structure. 
Of  animals,  only  the  chimpanzee  seems  to  be  susceptible. 

They  develop  in  the  general  lymphatic  system,  the  spleen  in  partic- 
ular, where  they  are  protected  from  the  bactericidal  power  of  the  blood. 
After  a  time,  however,  approximately  the  period  of  incubation,  they 
become  so  abundant  in  these  lymphatic  organs  that  they  are  carried 
over  into  the  general  circulation.  Then  as  a  result  of  bacteriolysis  the 
intracellular  toxins  are  liberated  and  symptoms  develop.  If  bacteri- 


TYPHOID  BACILLI  IN  THE  BLOOD 


121 


olysis  takes  place  other  than  in  the  blood  we  have  various  suppurative 
processes.  As  a  result  of  the  formation  of  antibodies,  the  development 
in  spleen,  etc.,  is  checked  but  should. these  immunity  reactions  become 
less  potent  relapses  may  occur  or  various  local  infections  manifest 
themselves. 

As  the  bacilli  do  not  multiply  to  any  extent  in  the  blood  itself  the  disease  cannot 
be  considered  as  a  typical  septicaemia  but  as  a  bacterisemia. 

Animals  are  not  susceptible  to  typhoid  fever  with  the  possible  exception  of  the 
higher  apes.  Of  course  the  injection  of  living  or  dead  cultures  may  kill  an  animal 
but  there  are  no  characteristic  localizing  symptoms. 


FIG.  *<;.— Bacillus  of  typhoid  fever,  stained  by  Loffler's  method  to  show  flagella. 

(Xiooo.)     (Williams.} 

Typhoid  bacilli  can  be  isolated  from  the  blood  during  the  latter 
period  of  incubation  and  rarely  after  the  tenth  day  of  the  disease.  It 
is  a  practical  point  that  the  time  to  isolate  the  bacteria  from  the  blood 
is  in  the  first  days  of  the  attack.  The  diagnosis  by  agglutination  is 
only  expected  after  the  seventh  to  tenth  day.  Agglutination  may  not 
appear  until  during  convalescence,  and  in  about  5%  of  the  cases  it  is 
absent.  It,  as  a  rule,  disappears  within  a  year. 

Very  little  success  has  been  obtained  with  curative  sera.  Chantamesse,  by 
treating  horses  with  a  nitrate  from  cultures  of  typhoid  bacilli  on  splenic  pulp  and 
human  defibrinated  blood,  claimed  to  have  obtained  a  curative  serum  possessing 


122  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

antitoxic  power.  Wright's  method  of  prophylactic  inoculation  is  now  being  em- 
ployed in  the  British  army  with  apparent  success.  In  this,  twenty-four-  to  forty- 
eight-hour-old  cultures  are  killed  at  53°C.;  K%  °f  lysol  is  then  added.  An  injec- 
tion of  500,000,000  bacteria  is  made  at  the  first  inoculation,  and  ten  days  later  an 
injection  of  1,000,000,000.  The  British  prefer  to  inject  subcutaneously  in  the  in- 
fraclavicular  region  and  at  the  insertion  of  the  deltoid.  The  Germans  consider  3 
injections  as  conferring  greater  immunity. 

Russell  has  obtained  splendid  results  in  the  U.  S.  Army  with  his  method  of 
vaccination.  In  this  3  injections  are  given  at  intervals  of  ten  days,  the  dosage 
being  500,000,000,  for  the  first  and  1,000,000,000  for  each  of  the  2  succeeding 
injections. 

Typhoid  vaccines  sterilized  with  0.5%  of  phenol  appear  to  keep  much  longer  and 
to  have  a  higher  immunizing  power  than  those  prepared  by  sterilization  with  heat 
and  subsequent  addition  of  the  antiseptic. 

Typhoid  bacilli  may  be  found  not  only  in  the  blood,  urine  and  faeces  but  as  well 
in  the  sputum  of  cases  showing  pulmonary  involvement.  They  have  also  been  found 
in  the  cerebrospinal  fluid  of  cases  showing  meningeal  symptoms.  At  the  autopsy 
they  may  be  found  in  the  spleen,  Pyer's  patches,  mesenteric  glands  and  liver. 

A  very  important  discovery  is  that  certain  persons,  who  may  have  had  only  a 
slight  febrile  attack,  may  eliminate  typhoid  bacilli  for  years  in  their  faeces  (typhoid 
carriers) .  The  bacilli  are  also  eliminated  for  considerable  periods  in  the  urine.  Dis- 
tinction is  now  being  made  between  acute  carriers  (convalescents)  and  chronic 
carriers. 

In  experiments  on  higher  apes  there  was  evidence  that  the  bacilli  eliminated  by 
carriers  are  in  many  instances  nonpathogenic.  About  one-half  of  typhoid  cases  are 
believed  to  be  due  to  contact  infections.  Drigalski  gives  it  for  Germany  as  64.7%. 
The  water  transmission  factor  is  of  less  importance  than  was  formerly  stated. 

The  most  satisfactory  method  of  detecting  carriers  is  by  examination 
of  faeces  or  urine  plated  out  on  Endo's  medium.  While  carriers  usually 
give  a  Widal  reaction  this  is  by  no  means  constant.  Typhoid  carriers 
are  said  to  maintain  a  high  opsonic  index. 

The  urine  and  faeces  of  typhoid  convalescents  should  be  proven  negative  by  cul- 
tural procedure  before  discharging  the  patients. 

Vaccination  may  possibly  be  a  satisfactory  measure  in  bringing  about  the  dis- 
appearance of  typhoid  bacilli  in  the  dejecta  of  carriers. 

For  laboratory  diagnosis,  blood  cultures  during  the  first  week  and 
agglutination  tests  during  the  second  week  and  onward  are  the  practical 
methods. 

Along  with  the  agglutination  tests  the  urine  and  faeces  should  be  cultured  on 
Endo's  plating  medium  and  later  transferred  to  Russell's  medium  for  cultural 
identification.  The  positive  identification,  provided  the  culture  so  isolated  shows  the 
cultural  characteristics  of  typhoid,  is  made  by  testing  the  bacilli  for  agglutination 
with  a  known  typhoid  serum.  Instead  of  the  usual  blood  cultures  one  may  use  the 


PARATYPHOID  123 

clot  in  the  Wright  U-tube  for  culturing  and  the  serum  remaining  after  centrifugaliza- 
tion  for  the  Widal  test  (clot  culture).  B.  typhosus  appears  in  the  blood  in  relapses. 
Kayser  considered  that  about  27%  of  cases  of  typhoid  in  Strasburg  were  caused  by 
raw  milk,  17%  by  contaminated  water,  17%  by  contact  with  typhoid,  and  10%  were 
due  to  typhoid  carriers.  Other  cases  were  due  to  infected  food,  and  about  13%  were 
of  origin  impossible  to  determine.  These  latter  may  have  been  due  to  unrecognized 
typhoid  carriers.  He  does  not  attach  the  same  importance  to  fly  dissemination  as 
do  American  authors. 

Contact  infection  is  the  great  factor  in  perpetuating  typhoid  fever 
but  this  agency  shows  diminishing  cases  each  year  provided  water  and 
milk  supplies  are  safe.  The  leading  European  cities  as  a  result  of  a 
safe  water  supply  rarely  show  more  than  about  3  typhoid  deaths  per 
100,000  population  per  year.  Edinburgh  shows  less  than  one  per 
200,000  for  the  year  1910.  In  American  cities  rates  of  12  to  15  per 
100,000  are  common. 

The  Gartner  or  Meat-poisoning  Group. — Under  this  designation 
may  be  considered  the  organisms  which  cause  gastrointestinal  disorders 
of  varying  degrees,  infection  with  which  is  usually  brought  about  by  the 
ingestion  of  meat  obtained  from  diseased  cattle.  Unless  the  meat  is 
thoroughly  cooked  the  bacilli  in  the  interior  may  not  be  killed. 

In  this  group  may  be  placed  B.  enteritidis,  the  typical  meat-poisoning  organism, 
B.  paratyphoid  B,  B.  Danysz,  B.  Aertryck,  B.  typhi  murium  and  B.  suipestifer. 

B.  suipestifer  or  the  hog  cholera  bacillus  was  formally  thought  to  be  the  cause  of 
this  important  epizootic.  It  is  found  in  the  intestines  of  quite  a  percentage  of 
healthy  hogs.  The  cause  is  now  known  to  be  a  filterable  virus. 

These  organisms  are  alike  morphologically  and  culturally  and  show  quite  a 
tendency  to  bipolar  staining  and  reduction  of  neutral  red  with  fluorescence  in 
forty-eight  hours.  B.  paratyphoid  B,  B.  Aertryck  and  B.  suipestifer  are  alike  from 
an  agglutination  standpoint,  while  B.  enteritidis  and  B.  Danysz  show  similarity 
in  this  respect.  B.  paratyphoid  A  stands  by  itself. 

Paratyphoid  Bacilli  (Achard  and  Bensaude,  1896;  Schottmiiller, 
1901). — Cases  resembling  mild  attacks  of  typhoid  occasionally  show 
agglutination  for  paratyphoid  bacilli.  These  organisms  have  also  been 
isolated  from  the  blood,  as  with  typhoid.  Two  types  have  been  recog- 
nized: the  paratyphoid  A.  and  the  paratyphoid  B.  The  latter  occurs 
in  80%  of  such  cases.  Culturally,  paratyphoid  B.  cannot  be  separated 
from  Gartner's  bacillus.  In  paratyphoid  A.  there  is  less  gas  produced 
in  glucose  bouillon  than  with  paratyphoid  B.,  and  the  primary  acidity 
of  litmus  milk  is  not  succeeded  by  a  subsequent  alkalinity.  It  does 
not  seem  practical  to  draw  a  fine  distinction  between  these  two  strains. 


124  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

Paratyphoid  B.  not  only  gives  symptoms  resembling  a  mild  typhoid  infection, 
but  may  show  symptoms  more  like  those  of  meat  poisoning  or  even  cholerine.  It 
is  more  pathogenic  for  laboratory  animals  than  is  B.  typhosus.  The  development  of 
antibodies  upon  immunizing  a  man  or  animal  with  paratyphoid  organisms  does  not 
seem  to  approach  that  obtained  with  typhoid. 

Castellani  has  conducted  experiments  with  typhoid  and  paratyphoid  vaccines 
and  has  found  that  typhoid  vaccines  give  an  agglutinating  serum  of  about  i  to  350 
titre  from  the  second  to  fifth  week  dropping  to  about  i  to  100  after  three  or  four 
months.  Paratyphoid  A.  gives  one  of  about  i  to  75  for  the  first  month  which  drops 
to  about  i  to  60  after  four  months.  Paratyphoid  B.  gives  about  one-half  the 
agglutination  response  that  paratyphoid  A.  does. 

Bacillus  Enteritidis  (Gartner,  1888). — This  organism  has  been  fre- 
quently isolated  from  cases  of  gastroenteritis  from  ingestion  of  infected 
meat. 

Meat  from  healthy  animals  which  has  been  in  contact  with  that  of  diseased 
animals  may  become  infected.  The  simple  act  of  placing  a  piece  of  infected  meat 
on  a  sound  piece  may  infect  the  latter.  It  has  been  noted  that  the  bacteria,  or  their 
toxins,  may  be  distributed  unevenly  in  the  meat  eaten,  so  that  one  person  consuming 
the  same  meat  may  be  made  very  ill  while  others  eating  this  meat  may  escape 
infection.  Infection  of  food  may  occur  not  only  from  unclean  handling  but  from  the 
material  carried  by  flies  or  even  from  the  faeces  of  mice  or  rats  deposited  on 
foodstuffs. 

This  organism  is  very  pathogenic  for  laboratory  animals,  producing  a  haemor- 
rhagic  enteritis  and  at  times  a  septicaemia.  Where  meat  has  been  contaminated  with 
Gartner's  bacillus  toxins  may  have  been  produced,  and  symptoms  of  poisoning  with 
acute  gastroenteritis  would  occur  shortly  after  ingestion.  This  is  not  a  true  toxin 
as  it  does  not  require  a  period  of  incubation  before  manifesting  its  toxic  action.  It 
is  interesting  to  note  that  this  toxin  is  not  destroyed  by  the  boiling  temperature,  thus 
differing  from  the  toxin  of  the  other  important  meat-poisoning  (botulism)  bacillus — 
B.  botulinus — which  is  rendered  innocuous  by  a  temperature  of  65°  or  7o°C.  If 
there  is  only  a  little  toxin  introduced  with  the  contaminated  meat,  the  symptoms 
will  be  delayed  one  or  two  days.  Such  organisms  have  been  isolated  in  pure  culture 
from  cases  with  high  fever,  marked  intestinal  derangement,  with  considerable  blood 
in  the  rather  fluid  stools.  In  two  cases  studied  the  disease  was  at  first  diagnosed 
as  a  severe  typhoid  infection.  Klein  thinks  the  organism  of  Danysz's  virus  (to 
kill  rats  during  plague  epidemics)  may  be  identical  with  B.  enter  itidis. 

Proteus  Vulgaris. — This  organism  is  often  encountered  in  plates 
made  from  faeces,  or  sewage  contaminated  water. 

It  is  common  in  decaying  meat  or  cheese,  and  cases  of  even  fatal  poisoning 
with  marked  gastrointestinal  symptoms  and  cardiac  failure  have  been  reported. 
At  times  it  is  the  cause  of  cystitis.  The  colonies  on  agar  are  moist  and  unevenly 
spreading  (amoeboid).  The  bacilli  are  very  motile,  long  and  slender,  tend  to  form 
filaments  and,  as  a  rule,  are  Gram-negative.  It  digests  blood-serum  and  is  a  rapid 
liquefier  of  gelatin.  In  litmus  milk  it  coagulates  with  a  soft  clot  and  an  alkaline 


DYSENTERY 


125 


reaction.  Subsequently  the  litmus  is  reduced  and  the  clot  digested  giving  a  dirty 
yellowish-brown  fluid.  Indol  is  rarely  produced.  The  cultures  generally  have  a 
putrefactive  odor.  In  infective  jaundice  (Weil's  disease)  this  organism  has  been 
reported  as  the  cause.  Organisms  of  this  group  were  formerly  designated  as  B. 
termo. 

Bacillus  Dysenterise  (Shiga,  1898). — Dysentery  bacilli  produce  a 
coagulation  necrosis  of  the  mucous  membrane  of  the  large  intestine 
and  occasionally  of  the  lower  part  of  of  the  ileum.  Polymorphonu- 
clears  are  contained  in  the  fibrin  exudate. 

It  was  formerly  thought  that  these  lesions  were  of  local  origin,  but  the  present 
view  is  that  toxins  are  produced  which,  being  absorbed,  are  eliminated  by  the 
large  intestine  with  resulting  necrosis.  Flexner,  by  injecting  rabbits  intravenously 
with  a  toxic  autolysate,  produced  characteristic  intestinal  lesions.  The  toxin  with- 
stands a  temperature  of  7o°C.  without  being  destroyed.  The  toxin  may  cause 
joint  trouble. 

There  are  two  main  types  of  dysentery  bacilli: 

1.  Those  producing  acid  in  mannite  media — the  acid  strains  (Flex- 
ner-Strong  types). 

2.  Those   not   developing   acid   in   mannite    (Shiga-Kruse   types). 
Ohno  finds  that  fermentative  reactions  do  not  correspond  to  immunity 
ones.     Thus  an  acid  strain  used  to  immunize  a  horse  may  produce  a 
serum  more  specific  for  a  nonacid  strain.     Hiss,  however,  found  that 
organisms  similar  in  fermentation  reactions  agreed  in  agglutination 
ones.     The  Shiga  type  is  very  toxic  in  cultures,  while  the  Flexner  type 
does  not  seem  to  possess  a  soluble  toxin. 

Clinically  the  toxaemia  of  cases  of  dysentery  due  to  Shiga  types  is  marked  while 
that  from  Flexner  strains  is  slight. 

We  often  designate  the  Shiga  strains  as  the  toxic  group  and  the  acid-producing 
strains  as  the  nontoxic.  The  "Y"  type  of  organisms  also  produces  acid  in  mannite. 
The  following  is  the  Lentz  table: 

Lentz  recognizes  4  types  of  dysentery  bacilli  for  the  differentiation  of  which  he 
uses  mannite,  maltose  and  saccharose  bouillon  with  litmus  as  an  indicator. 


Shiga-Kruse 

Flexner 

Strong 

«  Y" 

Mannite                

Blue 

Red 

Red 

Red 

Maltose 

Blue 

Red 

Blue 

Blue 

Saccharose  

Blue 

Blue 

Red 

Blue 

A  strain  which  ferments  not  only  mannite,  dextrose,  maltose  and  saccharose, 
but  dextrin  as  well,  is  known  as  the  Harris  type. 


126  STUDY   AND   IDENTIFICATION  OF  BACTERIA 

The  Shiga  strains  are  apt  to  cause  a  paresis  of  the  hind  extremities  of  the  injected 
rabbit  which  may  be  followed  by^  paralysis  and  death.  At  the  Lister  Institute 
injections  of  a  soluble  toxin  produced  a  serum  of  marked  antitoxic  power.  Such  a 
dysentery  serum,  which  is  probably  both  antitoxic  and  antimicrobic,  is  of  curative 
value.  Shiga  immunized  horses  with  polyvalent  cultures  and  obtained  a  polyvalent 
serum  which  has  reduced  the  death  rate  about  one-third. 

The  dysentery  bacillus  is  present  in  the  milky  white,  leukocyte  filled 
blood  flecked  mucous  stools  during  the  first  five  or  six  days  of  the  dis- 
ease. By  the  tenth  day  it  has  probably  disappeared.  Lactose  litmus 
agar  is  the  most  satisfactory  plating  medium.  The  stool  of  the  first 
two  days  may  give  practically  a  pure  culture.  The  staining  of  a  smear 
from  the  muco-purulent  stool  is  rich  in  phagocytic  cells,  many  of  them 
packed  with  Gram-negative  bacilli.  In  all  cultural  respects  the  dysen- 
tery bacillus  resembles  the  typhoid,  and  the  only  practical  method  of 
distinguishing  these  two  organisms,  other  than  by  agglutination  reac- 
tions, is  by  the  nonmotility  or  exceedingly  slight  motility  of  the  dysen- 
tery bacillus. 

The  characteristic  of  nonmotility  is  of  greatest  differentiating  value  and  the 
reports  of  slight  motility  are  probably  from  misinterpretation  of  molecular  movement 
as  motility.  The  dysentery  bacilli  do  not  form  those  threads  or  whip-like  filaments 
so  characteristic  of  typhoid  cultures  and  are  somewhat  plumper. 

As  a  rule  they  occur  singly  or  in  pairs,  and  of  rather  oval  or  even  coc- 
coid  shape  and  may  stain  bipolarly.  The  colonies  are  much  like  those 
of  typhoid.  Type  "Y"  colonies  often  show  indentation  while  the 
Shiga  types  show  round  colonies. 

The  dysentery  bacillus  is  not  found  in  the  blood  and  hence  is  not  eliminated  in 
the  urine.  It  is  found  in  mesenteric  glands.  In  dysentery  patients  agglutination 
phenomena  do  not  show  themselves  until  about  the  twelfth  day  from  the  onset. 
Hence,  this  procedure  is  of  no  particular  value  in  diagnosis.  It  is  of  value,  however, 
to  identify  an  organism  isolated  from  the  stools  at  the  commencement  of  the  at- 
tack, using  serum  from  an  immunized  animal  or  a  human  convalescent  for  the 
agglutination  test. 

Butler  has  suggested  taking  serum  from  dysentery  convalescents,  noting  the 
strain  involved,  and  preserving  it  by  taking  up  with  filter-paper  as  recommended 
by  Noguchi  for  the  Wassermann  haemolytic  amboceptor.  This  I  consider  very 
valuable  as  it  is  very  difficult  to  immunize  rabbits  with  a  Shiga  strain  on  account 
of  its  great  toxicity.  Dean  has  reduced  the  toxicity  of  Shiga  vaccines  by  treat- 
ing with  eusol. 

There  seems  to  be  very  little  agglutination  power  in  the  serum  of  convalescents 
from  Shiga  strains.  Flexner  strains  give  agglutination,  but  early  in  convalescence 
the  serum  is  not  apt  to  have  a  titre  of  more  than  i  to  50. 


COLON  BACILLUS 


127 


Morgan  has  reported  as  the  cause  of  certain  cases  of  bacillary  dysen- 
tery a  bacillus  known  as  B.  Morgan,  No.  i.  It  is  motile,  produce  indol, 
and  in  glucose  bouillon  gives  a  very  slight  amount  of  gas. 

It  does  not  change  mannite  and  does  not  produce  a  primary  acidity  in  litmus 
milk.  This  organism  is  a  frequent  cause  in  England  of  summer  diarrhoea  of 
children.  Flies  from  houses  with  such  cases  often  show  Morgan's  bacillus.  A 
dysentery  type  much  like  the  Flexner-Strong  strain  is  often  found  in  the  enteric 
affections  of  children  in  the  United  States. 

In  Japan,  dysentery-like  epidemics  of  a  very  fatal  disease,  termed 
ekiri,  occur  among  young  children.  The  organism  is  very  motile,  pro- 
ducing gas  and  acid  in  glucose  but  not  in  lactose  media.  It  is  reported 
at  times  to  show  indol  production.  Apparently  a  member  of  the  Gart- 
ner group. 

More  recently  a  strain  of  dysentery  bacilli,  known  as  Type  Y,  has  been  con- 
sidered of  importance.  This  organism  is  very  closely  related  to  the  Flexner  strain 
and  only  differs  from  it  in  that  it  requires  about  forty-eight  hours  to  turn  mannite 
litmus  media  pink  and  that  maltose  litmus  remains  blue.  An  organism  showing 
similar  cultural  characteristics  has  been  recently  recovered  from  faeces  of  laboratory 
rabbits  by  German  workers  investigating  the  problem  of  whether  certain  animals 
might  serve  as  carriers  for  dysentery. 

B.  COLI,  B.  LACTIS  AEROGENES,  B.  CLOACA 

While  the  COLON  BACILLUS  chiefly  inhabits  the  large  intestine,  the  B. 
lactis  aerogenes  is  to  be  found  in  the  upper  part  of  the  small  intestine. 
While  they  may  be  separated  on  the  ground  of  motility,  yet  it  is  by  the 
greater  fermentative  activity  of  the  B.  lactis  aerogenes  that  they  are 
best  separated.  Some  consider  them  as  only  representing  different 
strains  of  the  same  organism. 

The  main  points  of  distinction  are  gas  production  in  starch  media  (gas  bubbles 
on  potato  slant)  and  frequent  nonproduction  of  indol.  B.  lactis  aerogenes  is  closely 
related  to  the  pneumobacillus  and  at  times  shows  capsules.  It  is  best  differentiated 
from  the  pneumobacillus  by  its  gas  production  in  lactose  and  coagulation  of  milk. 

Some  consider  that  the  B.  coli  produces  a  bactericidal  substance  which 
inhibits  the  growth  of,  or  destroys,  pathogenic  bacteria  which  may  have 
passed  the  destructive  influences  of  the  gastric  juice;  others  that  this 
effect  is  due  to  their  free  growth  and  the  development  of  phenol  and 
various  putrefactive  substances. 

The  probable  importance  of  the  colon  bacillus  in  protecting  the  ^organism  is 
shown  by  the  fact  that  where  numerous  colonies  of  pathogenic  organisms  may  be 


128  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

cultivated  from  faeces  we  may  find  a  diminution  in  number  or  absence  of  the  colon 
bacillus.  This  condition  may  be  observed  in  infections  with  the  organisms  of 
dysentery,  cholera,  typhoid,  and  paratyphoid.  While  its  normal  function  is  prob- 
ably protective,  yet  the  B.  coli  is  an  important  pathogenic  agent,  it  being  fre- 
quently the  organism  isolated  from  purulent  conditions  within  the  abdominal  cavity, 
especially  in  appendicitis  and  lesions  about  the  bile  ducts.  It  is  particularly  prone 
to  cause  lesions  of  the  bladder  and  pelvis  of  the  kidney.  In  the  treatment  of  colon 
cystitis  by  vaccines  of  dead  colon  bacilli,  brilliant  results  in  opsonic  therapy  have 
been  obtained. 

Sir  A.  Wright  thinks  that  certain  cases  of  mucous  colitis  may  be  due  to  colon 
infection  and  that  vaccination  may  cure  them.  The  colon  bacillus  is  fully  con- 
sidered under  the  bacteriology  of  water. 

B.  cloaca  was  isolated  first  from  sewage  by  Jordan.  It  is,  as  a  rule, 
a  rapid  liquefier  of  gelatin,  and  in  its  reactions  with  sugars  and  litmus 
milk  resembles  the  colon  bacillus. 

Where  the  gelatin  liquefaction  is  slow  or  slight  B.  cloaca  may  be  dis- 
tinguished from  B.  coli  by  its  gas  formula  which  is  about  three  times  as 
much  CO2  as  H,  just  the  reverse  of  that  of  the  colon  bacillus.  B.  lactis 
aerogenes  is  also  often  found  in  sewage.  It  is  one  of  the  causes  of  the 
souring  of  milk. 

B.   ACIDOPHILUS,    B.    BlFIDUS,    B.    BULGARICUS 

These  are  often  termed  the  long  rod  group  of  lactic  acid  bacteria  in 
contradistinction  to  certain  other  Gram-positive  bacilli  which  are  short 
and  oval  and  which  are  confused  with  the  so-called  milk  streptococci. 

The  long  rod  group  often  forms  chains  and  often  shows  metachromatic  granules 
which  stain  with  Neisser's  method.  They  are  readily  distinguished  from  Gram- 
negative  lactic  acid  producers,  of  which  the  type  is  B.  lactis  aerogenes,  by  their 
Gram-positive  staining.  B.  acidophilus  often  give  the  impression  of  a  diphtheroid 
in  a  Gram  stained  faeces  smear.  It  is  nonmotile  and  often  shows  polar  granules. 
Grows  only  at  temperatures  above  22°C.,  op.  37°C.  It  grows  better  anaerobically 
than  aerobically  and  then  shows  the  clubbed  involution  characteristics  of  B.  bijidus; 
so  that  some  consider  these  organisms  the  same,  the  morphology  of  B.  bijidus  being 
the  result  of  anaerobiosis.  Original  cultures  are  best  made  in  i%  glucose  and  i% 
acetic  acid  bouillon.  Some  authorities  consider  B.  bijidus  the  most  important 
representative  of  the  large  intestine  flora.  B.  lactis  acidi  is  less  thermophilic  than 
B.  acidophilus  and  coagulates  milk  which  B.  acidophilus  does  not  do.  Certain 
polar  granule  bacteria,  as  B.  granulosum,  found  in  Yoghurt,  are  similar  to  B.  acido- 
philus but  coagulate  milk;  no  gas.  B.  bulgaricus  is  the  type  of  the  group  and  is 
discussed  under  milk. 

Rodella  thinks  B.  acidophilus,  B.  bijidus,  B.  gastrophilus  and  the  Boas-Oppler 
bacillus  identical.  B.  bulgaricus  is  said  to  never  show  polar  granules.  B.  bulgaricus 
and  the  group  of  organisms  similar  to  it  found  in  buttermilk,  etc.,  are  widely  used  in 


CHROMOGENS  I2Q 

the  treatment  of  various  intestinal  troubles.  North  has  used  cultures  of  B  bulgaricus 
for  extermination  of  undesirable  organisms  in  other  parts  of  the  body  than  the 
alimentary  canal  (used  as  applications  in  nasal,  throat  or  genito-urinary  infections). 

CHROMOGENIC  BACILLI 

These  are  identified  by  the  color  of  their  colonies  on  agar.  The 
B.  pyocyaneus  is  the  most  important  one  of  them  in  medicine,  but  the 
B.  prodigiosus  is  also  of  interest  medically.  A  violet  chromogen,  the 
B.  yiolaceus,  which  is  motile  and  liquefies  gelatin,  has  been  described 
under  many  names.  It  has  been  found  in  water. 

An  orange-yellow  chromogen,  the  B.  fulvus,  is  nonmotile  and  varies  as  to  its 
liquefaction  of  gelatin. 

B.  pyocyaneus  (Gessard,  1882).— This  organism  is  frequently  termed 
the  bacillus  of  green  or  blue  pus.  It  is  a  small  (2. 5X0.5/4)  motile 
Gram-negative  bacillus. 

It  is  generally  a  slender  delicate  bacillus  often  showing  thread-like 
arrangement  but  at  times  it  may  appear  as  short  plump  rods.  It  grows 
readily  at  room  or  incubator  temperature.  It  liquefies  gelatin  rapidly. 
The  green  color  diffuses  through  the  agar  or  gelatin  on  which  it  grows, 
so  that  we  not  only  have  the  green-colored  colony,  but  the  medium  as 
well  is  colored.  Upon  potato  the  colonies  are  more  of  a  deep  olive 
green  to  dirty  brown. 

No  gas  is  produced  in  either  glucose  or  lactose  bouillon;  blood-serum  is  digested, 
the  pitted  surface  showing  a  reddish-brown  color.  The  protein  ferment  pyocyanase 
has  been  used  to  remove  diphtheritic  membrane  and  for  treatment  of  M,  catarrhalis 
nasal  catarrhs.  There  are  2  pigments — a  green  water  soluble  one  and  a  blue 
one  soluble  in  chloroform. 

It  is  widely  distributed  in  water  and  air,  and  is  frequently  isolated 
from  faeces.  The  B.  fluorescens  liquefaciens  of  water  seems  to  be  simply 
a  strain  of  B.  pyocyaneus.  The  B.  pyocyaneus  is  frequently  associated 
with  other  pus  organisms  in  abdominal  abscesses. 

In  addition  to  having  an  endotoxin,  it  produces  a  soluble  toxin  similar  to  diph- 
theria toxin.  This  toxin  differs  from  those  of  diphtheria  and  tetanus  in  that  it  can 
stand  a  temperature  of  ioo°C.,  while  those  of  diphtheria  and  tetanus  are  destroyed 
at  about  65°C.  The  fact  that  the  union  between  toxin  and  antitoxin  is  only  of  a 
binding,  neutralizing  nature  is  best  shown  by  taking  a  mixture  of  pyocyaneus  toxin 
and  antitoxin  which  is  innocuous  and  heating  it.  This  destroys  the  antitoxin,  but 
does  not  injure  the  toxin.  We  now  find  that  the  original  toxicity  has  returned.  The 
antitoxins  of  diphtheria  and  tetanus  are  more  stable  than  the  corresponding  toxins; 
9 


130  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

hence,  this  experiment  would  be  impossible  with  them,  as  upon  heating  we  should 
first  destroy  the  toxin. 

On  account  of  the  frequent  association  of  B.  pyocyaneus  with  other 
organisms  of  better  recognized  pathogenicity  it  has  until  more  recently 
been  considered  rather  harmless;  this  view  can  no  longer  be  entertained 
as  it  is  frequently  the  sole  cause  of  middle-ear  inflammations,  intestinal 
disorders,  cystitis  and  possibly  at  times  of  septicaemia. 

B.  Prodigiosus. — This  is  a  very  small  coccobacillus  which  shows  mo- 
tility  in  young  bouillon  cultures.  It  is  Gram-negative.  The  colonies 
on  agar  or  other  solid  media  show  a  rich  red  color.  The  pigment  only 


FIG.  36. — Bacillus  pyocyaneus.     (Kolle  and  Wassermann.) 

develops  at  room  temperature,  it  is  absent  in  cultures  taken  out  of  the 
incubator.  The  B.  prodigiosus  is  frequently  found  on  foodstuffs,  espe- 
cially bread,  where  it  may  simulate  blood.  It  liquefies  gelatin  rapidly 
and  gives  a  diffuse  turbidity  to  bouillon.  It  is  probable  that  B.  indicus 
and  B.  kilensis  are  strains  of  B.  prodigiosus. 

Coley's  fluid,  which  has  been  used  in  cases  of  inoperable  sarcoma  and  other 
malignant  growths,  is  a  culture  prepared  by  growing  very  virulent  streptococci  in 
bouillon  for  ten  days.  This  Streptococcus  culture  is  now  inoculated  with  B.  prodi- 
giosus, and  after  another  ten  days  the  mixed  culture  is  killed  by  heat  at  60° C.  and 
the  sterile  product  injected.  Coley  injected  about  J^o  c.c.  of  this  vaccine.  At 
present  he  uses  nonfiltered,  heat  sterilized  bouillon  cultures  of  a  Streptococcus 
obtained  either  from  a  case  of  erysipelas  or  septicaemia.  To  this  is  added  material 
from  agar  cultures  of  B.  prodigiosus,  grown  separately  and  sterilized  before  adding 
to  the  sterilized  streptococcus  bouillon  culture. 


CHAPTER  IX 

STUDY  AND   IDENTIFICATION   OF  BACTERIA.    SPIRILLA. 
KEY  AND  NOTES 

KEY  to  recognition  of  gelatin  liquefying,  motile  and  Gram-negative 
spiral  or  comma-shaped  organisms. 

A.  Do  not  give  the  nitroso-indol  reaction  with  sulphuric  acid  alone  in  twenty-four 
hours. 

1.  Produce  an  abundant  moist  cream-colored  growth  on  potato  at  room  tempera- 
ture. 

(a)  Finkler  and  Prior's  spirillum  (Vibrio  proteus).  Liquefaction  of  gelatin 
very  rapid.  No  air-bubble  appearance  at  top  of  liquefied  area.  Cultures 
have  foul  odor.  Milk  coagulated.  Thicker  spirillum  than  cholera.  Iso- 
lated from  cholera  nostras. 

2.  Scanty  growth  or  none  at  all  on  potato  at  room  temperature.    Only  a  mod- 
erate yellowish  growth  when  incubated  about  incubator  temperature. 

(a)  Spirillum  tyrogenum  (Deneke's  spirillum).  Does  not  liquefy  gelatin  so 
rapidly  as  Finkler  Prior.  Thinner  and  smaller  spirillum  than  cholera. 

B.  Give  the  nitroso-indol  reaction  with  sulphuric  acid  within  twenty-four  hours. 

1.  Very  pathogenic  for  pigeons. 

(a)  Spirillum  metschnikovi.  Liquefies  gelatin  about  twice  as  rapidly  as 
cholera.  Gives  bubble  appearance  at  top  of  stab. 

2.  Scarcely  pathogenic  for  pigeons. 
(a)  Spirillum  cholerae  asiaticae. 

Nonmotile,  nonliquefying  and  Gram-positive  spirilla  have  also  been  described. 
There  is  also  a  large  group  of  phosphorescent  spirilla. 

Spirillum  Cholerae  Asiaticae  (Koch,  1884). — Typically,  the  morphol- 
ogy of  this  organism  is  that  of  the  comma  (Comma  bacillus  of  Koch). 
It  also  frequently  shows  S  shapes,  and  often  appears  in  long  threads 
showing  turns.  When  freshly  isolated  from  cholera  material  they,  as 
a  rule,  show  a  fairly  typical  morphology,  but  after  subcultures  in  the 
laboratory  variations  are  common,  so  that  rod  forms  and  round  in- 
volution shapes  give  a  picture  altogether  at  variance  with  the  comma 
shape. 

Even  in  recent  cultures  of  undoubted  cholera  we  may  have  different  types,  as 
coccoid  forms  and  slender  rods.  Ohno  has  noted  the  fact  that  the  same  strain  of 
cholera  will  give  at  one  time  vibrio  forms  and  again  coccoid  or  rod  forms,  depending 


132 


STUDY  AND   IDENTIFICATION   OF  BACTERIA 


on  the  reaction  of  the  media.  Inasmuch  as  the  recognition  of  vibrio  shapes  is  of 
importance  in  diagnosis  he  recommends  that  material  from  a  stool  be  inoculated 
into  3  tubes  of  peptone  solution  of  reaction  +0.3,  —0.5  and  —  i,  respectively,  one 
of  which  would  probably  show  vibrio  morphology. 


FIG.  37. — Cholera  spirilla.     (Kolle  and  Wassermann.) 

The  cholera  spirillum  is  very  motile  (a  scintillating  motility)  and 
liquefies  gelatin  fairly  rapidly,  although  more  slowly  than  any  of  the 
spirilla  mentioned  in  the  key. 


*~v 


FIG.  38. — Involution  forms  of  the  spirillum  of  cholera.     (Van  Ermengen.) 

The  colony  on  gelatin  was  formerly  considered  characteristic,  but  like  most 
cultural  characteristics,  it  is  now  considered  as  being  only  of  confirmatory  value;  it 
is  not  specific.  These  colonies  show  in  twenty-four  hours  as  small  granular  white 
spots  which  have  a  spinose  periphery.  An  encircling  ring  of  liquefaction  now 
makes  its  appearance  and  the  highly  refractile  (as  if  fragments  of  sparkling  glass) 
colony  can  be  separated  into  a  granular  center,  a  striated  periphery,  and  a  clear 
external  ring  of  liquefaction. 


CHOLERA 


133 


On  gelatin  stabs  the  liquefaction  produces  a  turnip-like  hollow  at  the  top  of  the 
puncture — the  air  bubble  appearance.  It  gives  the  nitroso-indol  reaction  with  sul- 
phuric acid  alone  (cholera  red).  Kraus  attaches  importance  to  the  fact  that  cholera 
does  not  produce  a  haemolytic  ring  on  blood  agar  as  do  the  pseudocholera  spirilla- 
a  difficulty  is  that  many  pseudospirilla  do  not  haemolize.  Furthermore,  true  cholera 
strains  may  occasionally  show  haemolysis,  especially  in  laboratory  cultures.  Quite 
a  discussion  has  arisen  in  connection  with  a  spirillum  isolated  from  cases  of  diarrhoea 
(no  symptoms  of  cholera)  in  pilgrims  at  El  Tor.  This  organism  gave  the  immunity 
reactions  (agglutination)  of  true  cholera  but  on  account  of  its  haemolytic  power  has 
been  considered  as  distinct  from  cholera.  Such  a  view  would  seem  to  be  untenable. 
Sp.  cholera  grows  very  rapidly  on  peptone  solution  and  this  is 
the  medium  for  the  enrichment  test  to  be  later  described.  On 
this  it  may  form  a  pellicle.  On  agar  the  colony  is  more  opales- 
cent (more  of  a  translucent  grayish  blue)  than  the  typhoid.  It 
does  not  grow  on  potato  except  at  incubator  temperature.  It 
does  not  coagulate  or  turn  acid  litmus  milk.  Some  strains, 
however,  do  produce  a  certain  amount  of  acid.  Using  the  Hiss 
serum  sugar  media  our  strains  produced  acid  in  glucose  and 
saccharose  but  not  in  lactose.  No  gas  production  in  any  of  the 
sugars.  The  spirilla  are  found  in  myriads  in  the  rice-water 
discharges,  these  white  flakes  being  desquamated  epithelial 
cells.  They  penetrate  the  crypts  of  Lieberkuhn,  but  rarely  ex- 
tend to  the  submucosa.  The  symptoms  are  due  to  an  endo- 
toxin. 

Cholera  may  be  transmitted  from  water  supplies, 
when  the  outbreak  is  apt  to  be  widespread  and  in 
great  numbers  from  the  start.     Also  by  indirect  con-       FlG     39._spi- 
tagion,  as  by  flies  or  on  lettuce,  etc.    A  very  im-    rillum  of  cholera 
portant  point  is  that  we  have  well  persons  whose    g^atin^two^day" 

fseces    contain   virulent    cholera    spirilla    (cholera    old.    (Fraenkel 

.  and  Pfeiffer.) 

carriers). 

Cholera  spirilla  disappear  from  the  stools  of  cholera  patients  very 
rapidly,  usually  in  five  to  ten  days. 

Only  exceptionally  are  organisms  excreted  longer  than  three  or  four  weeks,  but  cases 
are  on  record  of  periods  approximating  two  or  three  months.  Cholera  carriers  in  good 
health  may  come  down  with  cholera  as  the  result  of  administration  of  purgatives 
or  alimentary  canal  disorder.  This  would  explain  periods  of  incubation  longer 
than  the  usual  one  of  two  to  five  days. 

Cholera  carriers  are  therefore  of  less  importance  epidemiologically 
than  typhoid  carriers,  where  carrier  stage  may  last  years. 

It  is  well  to  remember,  however,  that  cases  have  been  reported  of  positive  findings 
after  a  period  approximating  two  months  from  the  onset  of  the  attack  of  cholera. 
Another  important  consideration  is  that  the  vibrios  may  be  absent  at  one  examina- 
tion and  be  present  at  a  later  one.  Purgatives  seem  to  influence  the  reappearance 


134  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

of  the  spirilla.  An  acid  reaction  of  the  faeces,  such  as  that  induced  by  lactic  acid 
bacteria,  would  apparently  be  of  value  in  the  prophylaxis  of  cholera  carriers. 

Greig  has  found  infection  of  the  bile  of  the  gall-bladder  or  ducts  in  80  cases  in 
271  cholera  autopsies.  While  cholera  spirilla  are  soon  crowded  out  by  intestinal 
bacteria,  thus  explaining  the  short  period  during  which  cholera  spirilla  are  excreted 
by  convalescents,  this  is  not  true  when  the  cholera  vibrio  gets  into  the  bile  ducts  or 
gall-bladder,  where  ideal  conditions  prevail  for  a  prolonged  life.  In  fact  bile  has 
recently  been  recommended  as  a  selective  medium  for  cholera  enrichment.  Greig 
found  one  cholera  convalescent  excreting  cholera  vibrios  forty-four  days  after  the 
attack.  Of  27  persons  who  had  been  in  contact  with  cholera  patients  6  were  ex- 
creting cholera  vibrios  though  apparently  well. 

To  identify  such  spirilla  immunity  reactions  are  necessary: 

1.  Injected  intraperitoneally  into  guinea-pigs,  it  produces  a  peritonitis  and  sub- 
normal temperature.     This  reaction  exists  for  spirilla  other  than  the  true  cholera 
spirillum. 

2.  Intramuscular  injections  into  pigeons  are  only  slightly  pathogenic,  if  at  all. 

3.  The  agglutination  test  is  the  most  practical.    In  this  we  use  serum  from  an 
immunized  animal,  in  dilution  of  from  100  to  1000.     It  is  rare  that  true  cholera 
vibrios  fail  to  agglutinate  in  serum  of  i  to  500  and  even  sera  of  i  to  10,000  dilution 
give  the  reaction.     Serum  of  cholera  convalescents  may  show  agglutination  as  early 
as  the  tenth  day;  it  is  usually  best  shown  about  the  third  week.     Dunbar's  quick 
method  is  very  practical.     Make  two  hanging-drop  preparations,  using  mucus  from 
the  stool  as  the  bacillary  emulsion.    To  one  add  an  equal  amount  of  a  i  :  50  normal 
serum;  to  the  other  a  i  :  500  dilution  of  immune  serum.     Cholera  spirilla  remain 
motile  in  the  control,  but  lose  motility  and  become  agglutinated  in  the  preparation 
with  the  immune  serum. 

4.  Pfeiffer's  phenomenon.    If  cholera  spirilla  are  introduced  into  the  peritoneal 
cavity  of  immunized  guinea-pigs  (or  if  together  with  a  i  :  1000  dilution  of  immune 
serum  the  mixture  is  injected  intraperitoneally  into  normal  guinea-pigs)  and  at 
periods  of  ten  to  sixty  minutes  after  injection,  material  is  removed  by  a  pipette  from 
the  peritoneal  cavity,  the  spirilla  have  lost  motility,  have  become  granular  and 
degenerated.     Pseudospirilla  are  unchanged.     This  reaction  may  be  carried  on  in 
a  pipette,  using  fresh  serum. 

Antisera  for  the  treatment  of  cholera  have  not  proved  successful. 

On  the  whole  the  reports  from  the  use  of  anticholera  sera  are  not 
very  encouraging.  Savas,  however,  was  favorably  impressed  by  such 
treatment  during  the  Balkan  war.  It  should  be  administered  intra- 
venously and  early  in  the  attack  and  given  in  doses  of  50  c.c.  Of  61 
severe  cases  so  treated  the  mortality  was  55.7%.  Of  17  severe  cases 
not  receiving  serum  treatment  all  died. 

Prophylactically,  there  are  three  prominent  methods:  i.  That  of  Haffkine,  where 
live  cholera  spirilla  are  injected  subcutaneously;  and  2.  Strong's  cholera  autolysate. 
In  this  cholera  cultures  are  killed  at  6o°C.  The  killed  culture  is  then  allowed  to 
digest  itself  in  the  incubator  at  37°C.  for  three  or  four  days  (peptonization).  The 
preparation  is  then  filtered  and  from  2  to  5  c.c.  of  the  filtrate  is  injected.  Ferran 
was  the  first  to  use  vaccines. 


DIAGNOSIS   OF   CHOLERA  135 

3.  At  present  the  same  methods  are  being  used  for  cholera  vaccines  as  for  those  of 
typhoid.  An  emulsion  of  the  vibrios  in  salt  solution  or  a  bouillon  culture  is  subjected 
to  a  temperature  of  S4°C.  for  one  hour.  Three  doses  are  injected  seven  to  ten  days 
apart,  going  from  500,000,000  to  2,000,000,000. 

Among  the  Greek  forces  0.45%  of  the  inoculated  and  1.9%  of  the  noninoculated 
were  attacked.  The  inoculated,  however,  were  sanitary  troops  and  hence  more 
exposed  to  infection. 

For  diagnosis:  i.  take  a  fleck  of  mucus,  make  a  straight  smear  and 
fix;  stain. with  a  i  :io  carbol  fuchsin.  The  comma-shaped  organisms 
appear  as  fish  swimming  in  a  stream. 

2.  Inoculate  a  tube  of  peptone  solution.     The  cholera  spirilla  grow 
so  rapidly,  and  being  strong  aerobes,  they  grow  on  the  surface  of  the 
fluid  so  that  by  taking  a  loopful  from  the  surface,  we  may  in  three  to 
eight  hours  obtain  a  pure  culture.     Should  there  be  a  pellicle  present, 
this  should  be  avoided  in  the  transfer  by  tilting  the  tube  slightly,  so  that 
the  material  near  the  surface  be  obtained  without  touching  the  pellicle. 
Inoculate  a  second  tube  from  the  surface  of  this  first  and,  if  necessary, 
a  third  (enrichment  method). 

3.  Test  for  cholera  red  reaction.     (Simply  adding  from  3   to  5 
drops  of  concentrated  chemically  pure  sulphuric  acid  to  the  first  or 
second  peptone  culture  after  eighteen  to  twenty-four  hours'  growth. 
Some  specimens  of  peptone  do  not  give  the  reaction.)     At  times  we 
only  get  the  cholera  red  when  we  have  a  pure  culture  of  cholera. 

4.  Smear  a  fleck  of  mucus  or,  better,  the  three-hour  surface  growth  of 
a  peptone  culture  on  a  dry  agar  surface  in  a  Petri  dish.    From  colonies 
developing,   make  agglutination  and,  if  desired,  cultural   tests.     It 
is  by  immunity  reactions  that  we  identify  cholera  spirilla.     The  surface 
moisture  of  plates  is  best  dried  by  the  filter-paper  top. 

The  cholera  colony  is  easily  distinguished  from  the  ordinary  faecal  bacterial  colonies 
by  its  transparent,  bluish-gray,  delicate  character.  It  emulsifies  with  the  greatest 
ease.  A  practical,  quick  method  is  to  make  smears  from  suspicious  colonies,  stain 
for  one  minute  with  dilute  carbol  fuchsin  and  if  vibrios  are  present  to  make  2 
vaseline  rings  on  a  single  slide  allowing  ample  space  at  one  end  for  handling  the 
preparation  safely.  Inside  of  one  ring  deposit  with  a  platinum  loop  a  drop  of  salt 
solution  and  inside  the  ring  nearest  the  end  which  is  to  be  held  by  fingers  or  forceps, 
deposit  a  loopful  of  i  to  500  or  i  to  1000  dilution  of  cholera  serum.  The  emulsion 
in  the  salt  solution  remains  uniformly  turbid  and  under  a  low  power  of  the  micro- 
scope (%  inch)  shows  a  scintillating  motility.  The  emulsion  made  into  the  drop 
of  serum  quickly  shows  a  curdy  agglutination  and  upon  examination  with  the 
%-inch  objective  shows  clumping  and  absence  of  motility.  ^  Cover-glasses  placed 
over  the  2  vaseline  rings  assist  in  the  study  of  the  preparation. 

Cholera  selective  media  are  considered  under  "Culture  Media." 


CHAPTER  X 


STUDY  AND  IDENTIFICATION  OF  MOULDS 

CLASSIFICATION  OF  THE  FUNGI 


Order 


Suborder  Family 


Phycomycetes      Zygomycetes  Mucoracidae 


Saccharo- 
mycetidae 


Gymnoas- 
cees 


Genus 
Mucor 
Rhizomucor 


Ascomycetes 


Is-' 
(s. 


Gymno- 
ascidae 


Carpoascees  Perisporiacidae 


Hyphomycetes 


Species 

M.  corymbifer 
M.  mucedo 
R.  parasiticus 
R.  niger 
cerevisiae 
anginas 
S.  blanchardi 
E.  vuillemini 
C.  gilchristi 

C.  hominis 

T.  sabouraudi 
T.  tonsurans 
T.  violaceum 
T.  mentagro- 
phytes 
T.  cruris 
M.  audouini 
A.  schoenleini 

{P.  montoyai 
P.  crustaceum 
S.  nidulans 
A.  fumigatus 
A.  concentricus 
A.  pictor 
A.  niger 

D.  bo  vis 
D.  madurae 
M.  mycetomi 
M.  furfur 

M.  minutissi- 
mus 

M.  albicans 
T.  giganteum 
S.  beurmanni 

NOTE. — In  many  of  the  works  on  bacteriology  considerable  space  is  given  to 
the  so-called  Higher  Bacteria.  The  organisms  are  chiefly  considered  under  the 
names  Leptothrix  or  forms  in  which  are  found  simple  nonbranching  threads,  Clado- 
thrix  or  thread-like  forms  with  false  branching  and  Streptothrix  or  forms  showing 
true  branching.  It  is  not  practical  to  consider  any  separate  group  distinct  from  the 
so-called  Lower  Bacteria  on  the  one  hand  and  the  Fungi  on  the  other. 

136 


Rhizopus 
Saccharomyces 

Endomyces 
Cryptococcus 


Trichophyton 


Microsporum 
Achorion 

Penicillium 
Sterigmatocystis 

Aspergillus 

Discomyces 

Madurella 
Malassezia 
Microsporoides 

Monilia 

Trichosporum 

Sporotrichum 


FUNGI  137 

The  Thallophyta  are  plants  in  which  there  is  no  differentiation  be- 
tween root  and  stem. 

The  classes  of  Thallophyta  which  are  of  interest  medically  are  i.  the 
Algae  and  2.  the  Fungi. 

The  Algae  contain  chlorophyll.  An  exception  to  this  is  with  the  Schizophyta, 
algae  which  include  the  Bacteriaceae  or  Schizomycetes  and  the  Schizophycese  or 
Cyanophyceae.  The  bacteria  do  not  contain  chlorophyll  and  the  Cyanophycese 
or  blue-green  algae  contain  a  blue  pigment  (phycocyanin)  in  addition  to  chlorophyll. 
It  is  in  their  relation  to  bacteria  that  algae  are  important.  Some  authorities  consider 
the  family  Bacteriaceae  as  belonging  to  the  order  Cyanophyceae. 

Diseases  caused  by  fungi  are  known  as  mycoses. 

Some  include  Lichenes  as  a  separate  class.  These  are  really  sym- 
biotic organisms — Fungi  parasitic  on  Algae. 

The  fungi  do  not  possess  chlorophyll.  They  are  in  their  simplest  forms  ramifying 
filaments  called  hyphae.  The  vegetative  hyphae  which  intertwine  in  tangled  threads, 
as  a  support,  are  termed  the  mycelium,  while  those  which  project  upward  are  called 
the  aerial  hyphae  and  are  the  ones  which  bear  the  conidia  or  spores. 

The  aerial  hypha  which  carries  the  fruiting  organ  encasing  the  conidia  (sporan- 
gium) is  called  the  sporangiophore  and  the  more  or  less  rounded  termination  of  this 
hypha,  which  projects  into  the  sporangium,  is  called  the  columella. 

The  hypha  may  be  composed  of  one  cell  or  of  many  cells  separated  by  septa 
(septate). 

The  orders  of  the  class  Fungi  which  are  of  interest  medically  are: 
i.  the  Phy corny cetes;  2.  the  Ascomycetes;  3.  the  Hyphomycetes. 

Phycomycetes. — These  produce  a  copious  network-like  mycelium,  which  is  non- 
septate,  and  reproduce  asexually  by  means  of  a  sporangium,  a  case-like  structure 
borne  on  the  clubbed  extremity  of  an  erect  hypha  (columella)  and  containing  numer- 
ous spores  or,  as  in  the  case  of  the  suborder  Oomycetes,  reproduction  is  by  heter- 
ogamy.  (Dissimilar  sexual  cells — a  smaller  male,  antheridium,  and  a  larger  female, 
oogonium.  By  fertilization  by  antherozoids  from  the  antheridium  penetrating  the 
oosphere  we  have  oospores.) 

The  suborder  Zygomycetes  reproduces  either  asexually  (a  sporangium  filled  with 
spores)  or  by  isogamy  (two  similar  but  sexually  differentiated  cells  conjugate  and 
form  on  fusion  a  zygospore). 

Belonging  to  this  suborder  we  have  four  families,  only  one  of  which,  the  Mucor- 
acidae,  is  of  importance  medically.  In  this  family  we  have  three  genera:  Mucor, 
without  rhizoids;  Rhhopus,  with  rhizoids  and  unb ranched  aerial  hyphae  and, 
Rhizomucor,  with  rhizoids  and  ramified  mycelium. 

Under  anaerobic  conditions  the  non-septate  mycelium  of  these  fungi  may  break  up 
into  short  septa  resembling  yeasts. 

Two  species  of  Mucor  are  of  pathogenic  importance. 


STUDY   AND   IDENTIFICATION   OF   MOULDS 

i.  Mucor  mucedo  and  2.  Mucor  corymbifer.  These  moulds  develop  especially  in 
external  cavities  as  nasopharynx  and  external  ear. 

Pulmonary  and  generalized  infections  have  also  been  reported.  The  pathogenic 
species  have  smaller  spores  and  grow  best  at  37°C.  The  thick,  coarse,  cotton-like 
mould  seen  on  horse  manure  is  a  Mucor.  The  sporangium,  the  organ  of  fructifica- 
tion, contains  the  spores  within  its  interior.  The  M.  mucedo  has  thick  silver-gray 
mycelium,  with  large  sporangia,  150/1  in  diameter,  containing  oval  spores,  5  X  QM- 
The  M .  corymbifer,  which  has  been  reported  from  a  generalized  infection,  considered 
as  typhoid,  shows  a  snow-white  mycelium.  The  sporangia  are  20  to  40ju  and  the 
spore  about  3/1  in  diameter. 

Rhizomucor  parasiticus  has  been  reported  from  the  sputum  of  a 
woman  with  a  condition  resembling  phthisis. 

Rhizopus  niger  has  a  columella  which  becomes  distorted  into  a  mushroom  shape 
after  the  spores  have  been  discharged  from  the  sporangium.  This  mould  has  been 
considered  as  the  cause  of  a  mycosis  of  the  tongue. 


>y 

FIG.  40: — Yeast  cells.     Saccharomyces  cerevisice.     (Coplin.) 

Ascomycetes. — In  this  order  are  included  many  of  the  parasitic 
moulds.  The  most  distinctive  characteristic  is  the  formation  of  asco- 
spores  in  an  ascus  (little  sac). 

It  is  an  enlargec!  extremity  of  a  hyphal  branch  in  which  a  definite  number  of  spores, 
usually  eight,  is  formed.  The  ascus  usually  ruptures  at  its  tip.  Other  members  of 
the  order  are  formed  from  hyphae  by  the  separation  of  cells  in  succession  from  the 
free  cells.  The  mycelium  is  septate. 

The  order  is  divided  into  those  with  naked  asci  (Gymnoascees)  and 
those  having  a  perithecium  or  investing  layer  about  the  ascus  (Car- 
poascees). 


YEASTS 


139 


Belonging  to  the  suborder  Gymnoascees  we  have  i.  the  family  of  Saccharomyce- 
tidas,  which  reproduce  by  budding  and  in  which  the  asci  are  without  any  semblance 
of  a  sheath,  and  2.  a  family  in  which  there  is  an  indication  of  the  formation  of  a  peri- 
thecium — this  may  be  termed  the  Gymnoascidae  family. 

SACCHAROMYCETID.E. — There  are  three  genera:  Saccharomyces,  Endomyces,  and 
Cryptococcus. 

Saccharomyces. — These  reproduce  by  budding,  have  ascopores  and  no  mycelial- 
like  threads. 

S.  eeremsia. — This  is  the  ordinary  yeast  fungus.     Used  at  times  as  an  antiseptic. 
It  is  also  used  in  treatment  of  beriberi  as  it  is  very  rich  in  vitamines. 
S.  angina. — Found  in  a  case  of  angina. 

S.  blanchardi. — Found  in  a  jelly-like  tumor  mass  of  the  abdomen.  The  budding 
cells  varied  from  2  to  20^1.  Probably  identical  with  S.  tumefaciens,  reported 
as  the  cause  of  a  subcutaneous  tumor  about  region  of  Scarpa's  triangle. 
Endomyces. — Forms  spores  in  the  interior  of  filaments,  or  by  ascus  formation  or  by 
chlamydospores  (resistant  spore-like  structures  with  a  thick  membrane  which 
project  from  the  extremities  or  sides  of  the  hyphae  as  bud-like  structures). 
E.  vuillemini. — One  of  the  organisms  of  thrush.  It  produces  a  false  membrane, 
especially  on  buccal  surfaces,  which  is  easily  detached  and  beneath  which  the 
mucosa  is  intact.  Grows  only  in  acid  media.  Hence  propriety  of  alkaline 
treatment.  Some  authorities  consider  the  genus  Endomyces  as  the  same  as 
Monilia  or  Oidium. 

Cryptococcus. — Reproduces  by  budding,  but  ascospore  formation  not  observed. 
Not  a  well-recognized  genus.  The  diseases  caused  by  it  are  termed  blasto- 
mycoses. 

C.  gilchristi. — The  cells  are  about  i6ju  in  diameter  and  have  a  thick,  double  con- 
toured membrane.  They  reproduce  by  budding.  The  skin  lesions  resemble 
various  infectious  granulomata  and  diagnosis  rests  on  the  finding  of  budding 
or  sporulating  cells.  It  may  invade  internal  organs.  Original  cultures  are 
obtained  with  some  difficulty  and  then  best  with  Lender's  serum.  Subcultures 
grow  readily.  Potato  is  a  good  medium  and  on  it  we  may  have  both  mycelial 
and  yeast-like  growth.  Guinea-pigs  can  be  inoculated  subcutaneously.  A  mould, 
somewhat  similar,  is  the  Coccidioides  immitis  of  Ophuls.  This  has  a  mycelial 
growth  in  tissues,  this  distinguishing  it  from  the  former  fungus.  The  large 
cells  frequently  show  yeast-like  bodies  within,  hence  the  characteristic  of  en- 
dogenous spore  formation.  The  large  round  encapsulated  cells  of  C.  gilchristi 
or  C.  hominis  do  not  show  contained  spores.  The  infection  frequently  becomes 
generalized.  The  small  bodies,  about  3/1,  in  the  Molluscum  contagiosum  cells 
are  thought  by  some  to  be  yeasts.  They  are  more  probably  artefacts. 
Plimmer's  bodies  in  cancer  cells  belong  in  this  group.  They  also  are  probably 
other  than  parasites. 

C.  lingua  pilose.— This  is  a  more  or  less  elongated  yeast-like  organism  and  sup- 
posed to  be  the  cause  of  black  tongue,  a  benign  affection  of  the  lingual  papillae. 

GYMNOASCIDAE. — Belonging  to  the  family  Gymnoascidae  we  have 
the  genera  Trichophyton,  Microsporum,  Achorion,  Endodermophyton 
and  Epidermophyton. 


140 


STUDY   AND    IDENTIFICATION   OF   MOULDS 


Trichophyton. — The  fungi  of  the  genus  Trichophyton  are  generally  known  as 
the  large-spored  ringworms.  The  spores  are  in  chains  and  may  be  inside  the  hair 
or  both  outside  and  inside.  Many  of  them  are  of  animal  origin,  especially  from 
the  horse  and  the  cat.  The  spores  are  from  5  to  i5/z. 

The  mycelium  is  greatly  segmented,  shows  simple  or  dichotomous  branching, 
and  produces  spores  within  the  mycelium. 

T.  tonsurans. — Gives  a  crater-like  culture  with  fine  marginal  rays.  Fungus  wholly 
inside  the  hair.  Causes  most  of  the  large-spored  scalp  ringworms  and  many 
body  cases. 


10. 


FIG.  41. — More  common  fungi,  i,  Culture  of  Achorion  schoenleini  (favus); 
2,  culture  of  Trichophyton  tonsurans;  3,  culture  of  Trichophyton  sabouraudi;  4, 
sporangium  of  Aspergillus;  5,  culture  of  Trichophyton  mcntagrophytes;  6,  culture 
of  Microsporum  audouini;  7,  mycelium  and  spores  of  Malassezia  furfur;  8,  Crypto- 
coccus  gilchristi;  9,  A  and  B,  sporangium  and  mycelium  of  Mucor  corymbifer;  10, 
Penicillium;  n,  Saccharomyces  tumefaciens;  12,  Discomyces  bovis. 

It  is  the  T.  megalosporum  endothrix  of  Sabouraud. 

The  short,  diseased  fragmented  hairs  are  mouldy  looking.  The  spores  are  5  to  6 
microns. 

T.  sabouraudi. — Has  a  heaped-up  festooned  sort  of  culture.  There  is  a  similar 
fungus  with  a  violet  culture.  These  cause  some  of  the  scalp  and  beard  ring- 
worms. 

It  is  easily  dissociated  in  a  2  or  3%  solution  of  caustic  potash  while  T.  tonsurans 
is  hard  to  break  up.  The  hairs  are  broken  off  close  to  the  skin,  hence  "black  dotted 
ringworm." 


TINEA  IMBRICATA 


141 


T.  mentagrophytes.—This  is  the  T.  megalsporon  endoectothrix  of  Sabouraud.  The 
external  spores  are  in  chains  or  in  short  mycelial  threads,  not  mosaics  of  spores, 
and  are  of  very  unequal  size  (3  to  15  microns). 

The  internal  spores  are  scarce  and  are  from  5  to  6  microns  in  diameter.  To  exam- 
ine pull  out  downy  hairs  from  the  periphery  of  the  lesion  rather  than  the  dead 
central  ones.  There  are  varieties  from  horse,  cat,  and  bird.  The  lesions  are  more 
inflammatory  than  those  of  the  endothrix  class.  Most  of  the  beard  and  body  ring- 
worms belong  to  this  group— very  few  scalp  cases.  The  lesions  are  often  of  a  pus- 
tular type.  The  cultures  are  finely  rayed. 

Some  give  yellow  cultures,  others  white  and  one  derived  from  birds  a  rose-colored 
culture. 

Microsporum  audouini. — This  is  the  so-called  small-spored  ring- 
worm and  is  a  very  common  and  highly  contagious  affection  of  the  scalp 
in  children  in  England  and  France;  less  so  in  other  countries. 

It  is  almost  never  seen  in  the  tropics.  It  almost  exclusively  affects  the  hairy 
scalp.  The  spores  are  2  to  3/4  in  diameter.  The  broken  stump  of  the  hair  is  char- 
acteristic. The  fungus  is  packed  as  a  mosaic  of  spores,  forming  a  white  sheath, 
chiefly  on  the  outside  of  the  hairs.  It  gives  a  downy-white  culture. 

Achorion  schoenleini  is  the  cause  of  favus.  The  cultures  are  rather 
wrinkled.  It  is  characterized  by  the  scutulum  or  favus  cup.  This  is  a 
sulphur-yellow  pea-sized  cup  with  a  central  lusterless  hair.  Affected 
hairs  may  not  show  a  cup.  Favus  is  not  so  contagious  as  ringworm. 
It  chiefly  affects  the  hairy  scalp,  but  may  also  invade  the  nails  and  even 
the  body. 

Microscopical  examination  shows  great  irregularity  of  spores  and  mycelium,  the 
latter  being  irregularly  disposed  and  of  varying  thickness  and  length  and  wavy 
instead  of  straight  as  in  Trichophyton.  There  is  also  the  greatest  irregularity  in 
the  refractile  favus  spores — they  are  gnarled  and  bizarre  shaped,  in  contrast  to  the 
regular  ovals  or  spheres  of  the  ringworm  fungus.  Cultures  show  ridges  or  con- 
volutions. 

Endodermophyton  concentricum.  Castellani  considers  this  as  the 
causative  fungus  of  tinea  imbricata  rather  than  Aspergillus  concentricus . 

It  was  formerly  supposed  that  the  causative  fungus  was  Aspergillus  concentricus 
but  Castellani  has  demonstrated  that  fungi  of  this  genus,  when  present,  are  merely 
accidental.  He  has  isolated  in  cultures  what  he  considers  the  causative  fungus, 
Endodermophyton  concentricum.  He  treated  scales  for  ten  minutes  with  absolute 
alcohol  and  then  placed  single  scales  in  a  series  of  tubes  of  maltose  bouillon.  The 
fungus  grows  between  the  rete  malpighii  and  the  external  epidermal  layers  forming 
a  network  of  mycelial  threads,  about  3  microns  broad. 

Another  fungus  cultured  from  tinea  imbricata  scales  is  Endodermophyton  indicum. 
Inoculation  of  this  organism  in  pure  culture  produced  the  disease. 

The  characteristics  of  the  genus  Endodermophyton  are  the  growth  of  a  mycelial 


142  STUDY   AND   IDENTIFICATION   OF   MOULDS 

network  between  the  rete  malpighii  and  the  superficial  epidermal  layers.     In  cul- 
tures only  mycelial  filaments  are  found,  there  are  no  conidia  bearing  hyphae. 
The  fungus  is  also  called  Trichophyton  concentricum. 

Epidermophyton  cruris.     (See  Microsporoides  minutissimus,  page  145) . 
Under  the  name  "dhobie  itch"  this  fungus  affection  is  probably 
better  known  to  Europeans  than  any  other  tropical  skin  disease. 

This  name  dhobie  or  washerman's  itch  has  been  given  on  account  of  associating 
it  with  the  infection  of  the  underclothing  while  being  washed  in  the  pools  or  streams 
along  with  the  garments  of  those  who  have  this  skin  disease.  This,  like  every  other 
widespread  view,  has  probably  some  foundation  but  cannot  be  verified.  It  is  the 
eczema  marginatum  of  Hebra. 

This  affection  is  caused  by  various  species  of  Epidermophyton.  This  genus 
differs  from  Trichophyton  in  that  it  never  invades  the  hair  or  hair  follicles. 

The  species  which  have  been  more  frequently  reported  are  Epidermophyton  crttris, 
E.  perneti  and  E.  rubrum.  The  mycelium  is  about  4  microns  broad  and  the  spores 
about  5  or  6  microns.  All  of  these  fungi  can  be  cultured  on  Sabouraud's  maltose 
agar,  growth  appearing  in  about  a  week,  except  E.  perneti,  which  grows  more  rapidly. 

IN  THE  SUBORDER  CARPOASCEES  we  have  to  consider  the  family 
Perisporiacidae.  In  this  family  the  asci  are  completely  inclosed  by  the 
investing  membrane,  the  perithecium.  When  this  rots  the  spores  are 
set  free.  There  are  three  genera  of  interest,  Penicillium,  Aspergillus 
and  Sterigmatocystis. 

In  Penicillium  we  have  vertical  branches  with  strings  of  conidia.  In  Aspergillus 
these  conidia  arise  from  a  globular  termination  of  the  hypha. 

Penicillium. — While  Penicillium  does  at  times  form  perithecia,  yet  they  char- 
acteristically show  chains  of  spores.  The  common  P.  glaucum  resembles  a  hand 
with  terminal  beads,  more  than  the  hair  pencil,  from  which  the  name  is  derived. 

P.  crustaceum. — Is  the  common  blue-green  mould.  It  has  been  deemed  patho- 
genic in  cases  of  chronic  catarrh  of  the  Eustachian  tube  and  in  gastric  hyper- 
acidity. 

P.  montoyai. — Cause  of  violet  pinta. 

Aspergillus. — These  have  sterigmata  carrying  chains  of  spores,  these  sterigmata 
being  little  processes  projecting  out  from  the  knob-like  termination  of  the  aerial 
hypha  (columella).  Of  the  pathogenic  Aspergilli  we  have: 

1.  A.  fumigatus. — This  has  been  considered  as  the  cause  of  pellagra.     A  pul- 
monary mycosis  resembling  phthisis  may  be  due  to  this  species. 

2.  A  repens. — This  has  been  found  in  the  auditory  canal  and  may  produce  a 
false  membrane. 

3.  A.  flavus. — This  has  been  found  in  the  discharges  of  chronic  ear  diseases. 

4.  A.  concentricus. — Formerly  considered  as  the  cause  of  an  important  tropical 
ringworm,  tinea  imbricata.     The  scales  are  dry,  like  pieces  of  tissue-paper. 
There  are  generally  about  four  rings  which  do  not  heal  in  the  center.     General 
appearance  is  that  of  watered  silk.     There  are  no  inflammatory  lesions.     Com- 


PINT  A  143 

mon  in  Malay  peninsula.     Also  found  in  some  parts  of  the  Philippines  and  in 
China.     Some  authorities  consider  the  fungus  to  be  a  Trichophyton. 
Castellani  claims  that  the  cause  is  a  fungus  which  develops  between  the  stratum 
corneum  and  the  deeper  layers  of  the  epidermis,  Endodermophyton  concentricum. 
It  differs  from  the  achorions  in  not  invading  hair  follicles.     (See  page  141.) 

5.  A.  pictor. — This  is  the  cause  of  a  skin  affection  of  Central  America  (Pinta). 
In  the  affection  colored  spots  appear  on  the  skin,  chiefly  on  face,  forearms,  and 
chest.  The  disease  is  attended  with  a  mangy  odor.  Spots  are  of  various 
colors;  if  the  superficial  epithelium  is  affected  we  have  a  dark  violet  color. 
Deeper  involvement  gives  red  spots. 

Other  names  for  the  disease  are  caraate  and  mal  de  los  pintos.  At  first  it  was 
thought  that  the  different  colors  shown  by  the  eruption  were  due  to  varying  depths 
of  the  proliferating  fungi  in  the  skin  layers  but  it  is  now  known  that  the  explanation 
is  in  a  variety  of  species  in  the  different  types  of  pinta. 

The  pure  violet  pinta  is  caused  by  Aspergillus  pictor,  while  the  grayish- violet  one 
is  due  to  Penicillium  montoyai.  A  species  of  Monilia  causes  the  white  variety  and 
different  species  of  Montoyella  a  black  and  a  red  variety  respectively.  The  genus 
Montoyella  is  stated  by  Castellani  to  have  both  slender  and  thick  mycelial  threads, 
from  the  thicker  of  which  spring  delicate  hyphae  terminating  in  pear-shaped  conidia. 
Material  scraped  from  the  lesions  and  mounted  in  liquor  potassae  shows  the 
fructification  terminations  characteristic  of  Aspergillus  or  Penicillium  in  the  violet 
or  gray- violet  varieties  while  the  white,  black  and  red  ones  only  show  mycelial  threads 
and  scattered  spores.  These  pinta  species  of  fungi  can  be  cultivated  on  Sabouraud's 
medium. 

Montoya  thinks  that  the  pinta  fungi  lead  a  saprophytic  existence  in  the  waters  of 
mines  or  other  places  with  a  constant  high  temperature,  and  states  that  he  has 
obtained  pure  cultures  from  such  sources. 

Sterigmatocystis. — This  genus  has  chains  of  conidia,  similiar  to  those 
of  Penicillium,  but  these  are  borne  on  other  short  chains,  which  arise 
from  the  clubbed  aerial  hyphae  (conidiophores).  These  are  called 
secondary  and  primary  sterigmata,  respectively. 

S.  nidulans. — This  fungus  has  been  found  in  cases  of  otomycosis  as  well  as  in  the 
white  granules  of  mycetoma. 

Hyphomycetes.— In  this  order  are  grouped  certain  genera  which 
cannot  properly  be  assigned  to  any  of  the  other  orders.  They  are  also 
designated  Fungi  Imperfecti,  for  the  reason  that  the  fruiting  bodies 
characteristic  of  the  other  orders  have  not  been  satisfactorily  observed. 

Discomyces  bo-vis  —  This  is  the  well-known  ray  fungus,  the  cause  of  actinomycosis.  In 
man  it  is  at  times  found  in  chronic  suppurative  conditions  attended  with  much 
granulation  tissue.  Such  pus  may  show  small  yellow-gray  granules  about  the 
size  of  a  pin's  head.  When  spread  out  between  two  slides  the  central  portion 
shows  a  network  of  mycelium  with  bulbous  thread-like  rays  going  to  the  periph- 
ery. The  "clubs"  at  the  periphery  are  degenerate  structures  and  do  not  stain 
by  Gram.  The  central  mycelium  is  Gram-positive.  This  mould  is  essentially 
an  anaerobe  and  should  be  cultivated  in  a  deep  glucose  agar  stab.  It  may  also 


144 


STUDY   AND   IDENTIFICATION   OF   MOULDS 


be  cultivated  in  bouillon.  In  this  it  grows  at  bottom.  Growth  is  dry  and 
chalky.  In  diagnosis  look  for  the  little  granules.  Curetting  of  the  sinuses 
may  give  the  "ray  fungus"  when  they  are  not  found  free  in  the  pus. 
Discomyces  madura. — This  is  a  ray  fungus  found  in  the  yellow  " fish-roe"  granules 
of  madura  foot.  The  disease  is  caused  by  the  penetration  of  certain  species  of 
fungi  into  the  tissues  of  the  foot,  although  rarely  the  hand  or  some  other  part  of 
the  body  may  be  affected.  These  species  of  fungus  develop  in  granulomatous 
areas  from  which  sinuses  lead  to  the  surface  of  the  foot,  in  the  discharges  from 
which  are  found  small  granules  resembling  those  found  in  the  discharges  from 
actinomycosis  lesions. 


££i?u^°n  (ft\  .Cndomyces     vuillemini 

(Q)(monilia   or  Oidium    albicans) 


Cpidermophyton     /y\  Coccidipid&s 
cruria  V«  I       immitta 


Cryptococcujs 

linyuaa-piloaoe.        QA    Trichosporum   giyanteum 


T.  imbricata   (aide  view) 
mycetorni 


homini,  ££%% 


FIG.  42. — Important  tropical  fungi. 

As  a  rule  only  one  kind  of  fungus  is  found  in  a  single  case.  The  most  common 
infection  is  that  due  to  Discomyces  madura  (Nocardia  madura)  which  is  the  fungus 
of  the  fish-roe  like  granules  of  the  pale  or  white  variety  of  mycetoma.  These  like 
the  fungus  of  actinomycosis,  Discomyces  bovis,  show  a  felted  mycelium  in  the  center 
and  peripheral  club-like  structures.  The  granules  are  yellowish  white  and  vary  in 
size  from  a  pin's  head  to  a  small  pea.  The  mycelial  threads  are  very  narrow,  i  to 
iH  microns.  It  grows  aerobically  and  the  cultures  show  slender  mycelial  threads 
which  are  Gram-positive.  This  is  the  organism  of  Carter's  white  mycetoma. 

Other  species  of  the  pale,  white  or  ochroid  group  of  mycetoma  fungi  are  Indiella 
mansoni  (Brumpt's  white  mycetoma)  Nocardia  asteroides  (Musgrave  and  Clegg's 
white  mycetoma)  Sterigmatocystis  nidulans  (Nicolle's  white  mycetoma)  and  several 
others. 


MONILIA  AND   SPRUE  145 

The  cases  caused  by  the  black  varieties  are  more  rare  and  are  characterized  by  the 
presence  in  the  discharge  from  the  sinuses  of  black  gunpowder-like  grains. 

These  hard,  brittle,  irregular  grains  are  caused  by  various  species  of  fungi  of 
which  the  best  known  is  Carter's  black  mycetoma  (Madurella  mycetomi).  This 
species  was  cultured  by  Wright  and  first  shows  a  grayish  growth,  later  becoming 
black.  Other  black  varieties  of  mycetoma  are  due  to  various  other  fungi.  Bouf- 
fard's  black  variety  is  caused  by  Aspergillus  bou/ardi.  DeBeurmann's  black  myce- 
toma has  as  cause  Sporotrichum  beurmanni. 

Besides  the  white  and  black  varieties  we  also  have  a  red  variety  of  mycetoma. 
The  fungus  grains  are  quite  small  and  reddish  in  color.     It  is  not  an  uncommon 
infection  in  certain  parts  of  Africa,  as  Senegal.     The  cause  is  Nocardia  pelletieri. 
Discomyces  carougeani  has  been  reported  as  the  cause  of  juxta-articular  nodes, 

but  Breinl  has  been  unable  to  verify  the  finding. 

M alassezia  furfur. — This  is  the  fungus  of  tinea  versicolor.  It  is  common  both  in 
temperate  and  in  tropical  climates.  It  is  characterized  by  dirty  yellow  spots 
about  covered  parts  of  the  body.  Scrapings  shows  a  profusion  of  mycelial 
threads  and  interspersed  spores.  It  is  very  difficult  to  cultivate.  The  organism 
usually  termed  the  bottle  bacillus  is  really  a  fungus  having  the  characteristics 
of  the  genus  Malassezia.  It  is  thought  to  be  the  cause  of  pityriasis  of  the 
scalp. 

Microsporoides  minutissimus. — This  is  generally  considered  as  the  cause  of  Ery- 
thrasma  or  dhobie  itch,  a  very  common  intertrigo  of  the  tropics.  It  is  char- 
acterized by  its  narrow  mycelium  and  small  spores.  Various  fungi  are  found 
in  this  affection.  Castellani  considers  the  chief  cause  of  dhobie  itch  to  be  a 
trichophyton.  Epidermo phyton  cruris. 

Clinically  this  affection  shows  festooned  areas  of  a  bright  red  color  which  tend 
to  clear  up  in  the  center  becoming  fawn  color.  As  a  result  of  the  intolerable  itching 
and  scratching  the  affection  tends  to  spread  from  its  favorite  sites — the  inner  sur- 
faces of  the  thighs  and  the  scrotum.  The  spores  and  mycelium  are  abundant  at 
the  onset  but  later,  one  may  not  find  any  evidence  of  the  mould.  In  some  of  the 
rapidly  spreading  cases  I  have  found  a  symbiosis  of  fungus  and  coccus,  the  bacterial 
elements  lying  packed  in  aggregations  scattered  through  the  mycelial  ground  work. 
Culturally  these  cocci  were  S.  pyogenes  aureus.  (See  page  142.) 

Monilia  albicans  (Oidium  albicans). — Castellani  separates  Monilia 
from  Endomyces  in  that  it  does  not  show  the  asci  and  internal  spores 
of  the  latter.  In  cultures  it  gives  budding  yeast-like  growths  and 
mycelial  threads.  On  Sabouraud's  medium  it  gives  a  whitish  growth. 
It  slowly  liquefies  gelatin  and  blood-serum  and,  after  acidifying,  clots 
milk.  It  is  recognized  as  the  organism  of  thrush.  Bahr  found  this 
fungus  in  the  deep  layers  of  the  tongue  as  well  as  in  the  cesophageal 
and  intestinal  coatings  of  sprue. 

Ashford  is  convinced  that  a  species  of  Monilia  which  he  is  sure  is  distinct  from 
M .  albicans,  is  the  cause  of  sprue.  He  states  that  this  fungus  is  common  in  the 
bread  of  Porto  Rico.  He  has  recovered  the  organism  from  sprue  lesions  and  has 


146 


STUDY  AND   IDENTIFICATION   OF   MOULDS 


produced  a  monilia  septicemia  in  rabbits  inoculated  with  the  sprue  fungus.     This 

fungus  is  quite  pathogenic  when  first  isolated  from  sprue  lesions. 

In  internal  organs  the  mycotic  areas  are  not  associated  with  pus  formation.     On 

two  occasions  he  has  produced  stomatitis  in  animals  by  feeding  experiments. 

Monilia  tropicalis. — Castellani  has  reported  this  fungus  as  the  cause  of  a  broncho- 
mycosis.  It  does  not  coagulate  milk  nor  liquefy  gelatin.  The  growth  on 
Sabouraud's  medium  is  mainly  of  yeast-like  cells. 

M .  Candida.  This  fungus  was  found  in  white  patches  on  tongue  of 
a  child.  The  conidia  are  7  to  8  microns  and  the  mycelium  i  to  ij^  mic- 
rons in  diameter. 

Boggs  has  recently  isolated  a  Monilia  which  he  considers  as  closely  related  to 
M .  Candida.  The  patient  was  at  first  thought  to  have  a  mammary  carcinoma  with 
axillary  gland  metastases.  Later  on  there  was  a  severe  cough  with  abundant  red- 


FIG.  43. — Thrush  fungus.     (Kolle  and  Wassermann.) 

dish-gray  sputum  which  showed  mycelium  and  yeast-like  cells.  Cultures  on  glucose 
agar,  potato,  etc.,  at  37°C.  and  at  room  temperature  gave  a  moist  glistening 
whitish  growth  which,  when  examined,  showed  only  yeast-like  cells,  no  mycelial 
growth.  Hyphae  only  showed  in  later  cultures. 

There  was  a  fair  growth  in  milk  with  after  three  or  four  weeks  an  alkaline  reac- 
tion and  firm  coagulum.  Slight  acid  production  in  glucose  but  none  in  lactose, 
saccharose  or  mannite. 

Mycelial  growth  was  more  rapid  in  anaerobic  cultures  than  in  aerobic  ones. 
Boggs  notes  that  his  Monilia  is  morphologically  indistinguishable  from  the  Monilia 
of  sprue.     The  buccal  mucosa  of  this  case  did  not  show  any  abnormalities. 
Trichosporum  giganteum. — This  is  the  cause  of  a  disease  of  the  hairs,  known  in 

Columbia  as  "Piedra,"  so  called  from  the  small  gritty-like  masses  along  the 

length  of  the  hair.     These  spores  are  arranged  like  mosaics  about  the  hair. 
Sporotrichum  beurmanni. — This  fungus  has  a  narrow  mycelium  (2/1)  and  branches 

in  all  directions.     The  spores  appear  as  little  grape-like  clusters  of  oval  spores 


SPOROTRICHOSIS  147 

(3  to  SM)  at  the  end  of  a  filament.     It  is  readily  cultivated,  showing  as  a  small 

white  growth  about  the  eighth  day. 

The  fungus  of  sporotrichosis  develops  in  tissue  by  budding,  not  showing  the 
mycelial  growth  seen  in  artificial  cultures.  Potato  makes  a  good  medium  and  often 
such  cultures  show  pigmentation. 

This  mould  produces  indolent,  glistening,  subcutaneous  tumors  which  are  pain- 
less. They  may  ulcerate  and  give  off  a  brownish  discharge.  They  resemble  tuber- 
culous or  syphilitic  lesions. 

Certain  organisms  which  resemble  both  moulds  and  bacteria,  having  branching 
filamentous  forms  and  at  the  same  time  having  a  spore-like  method  of  reproduction, 
are  known  under  the  names  Streptothrix  or  better  Nocardia.  It  is  chiefly  in  various 
pathological  processes  of  the  lungs  that  they  have  been  observed,  but  in  addition 
they  have  been  noted  in  brain,  glands,  kidney  and  subcutaneous  tissue. 

The  infections  are  most  likely  to  be  confused  with  phthisis  and  glanders.  The 
organisms  are  easily  cultivated  and  in  staining  reactions  are  midway  between  T.  B. 
and  Actinomycosis. 

Castellan!  uses  the  generic  name  Nocardia  for  Discomyces. 

DIAGNOSIS  or  FUNGI 

The  most  expeditious  way  to  examine  for  fungi  is  to  treat  the  scales 
or  hairs  with  a  10%  solution  of  caustic  potash  or  soda.  Then  crush 
between  two  slides;  heat  moderately  over  the  flame  and  examine. 

Tribondeau's  method  is  to  treat  the  scales  with  ether,  then  with  alcohol,  and 
finally  with  water.  Next  put  the  sediment  (it  is  convenient  to  use  a  centrifuge) 
in  a  drop  of  caustic  soda  solution.  Cover  with  a  cover-glass,  and  after  the  prepara- 
tion has  stood  about  an  hour  run  glycerine  under  the  cover-glass. 

A  very  satisfactory  method  is  to  scrape  the  scales  with  a  small  scalpel,  and  smear 
out  the  material  so  obtained  in  a  loopful  of  white  of  egg  or  blood-serum  on  a  glass 
slide.  By  scraping  vigorously  the  serum  may  be  obtained  from  the  patient.  After 
the  smear  has  dried,  treat  it  with  alcohol  and  ether  to  get  rid  of  the  fat.  It  may  then 
be  stained  with  Wright's  stain  or  by  Gram's  method.  The  ordinary  Gram  method 
may  be  used  or  the  decolorizing  may  be  done  with  aniline  oil,  observing  the  decolori- 
zation  under  the  low  power  of  the  microscope. 

Yeasts  are  best  examined  in  hanging  drop  on  the  plain  slide  with  vaselined  cell, 
as  given  under  Blood. 

An  excellent  way  to  examine  moulds  is  to  seize  some  of  the  projecting  sporangia 
from  the  surface  of  a  plate  with  forceps  and  mount  in  liquid  petrolatum.  I  have 
found  that  moulds  in  scales  from  skin  or  infecting  various  mites  or  insects  will  show 
a  growth  in  this  medium  when  mounted  on  a  slide  and  covered  with  a  cover-glass. 

CULTIVATION  or  FUNGI 

Moulds  grow  well  on  media  with  an  acid  reaction,  so  that  by  adjust- 
ing the  reaction  to  +2%  or  even  higher,  we  permit  of  the  growth  of  the 
fungi,  but  inhibit  bacterial  development. 


148 


STUDY   AND   IDENTIFICATION   OF   MOULDS 


Glycerine  agar,  bread  paste,  or  potato  media  are  all  suitable,  but  the  best  medium 
is  that  of  Sabouraud: 

Maltose,  4 .  o  grams. 

Peptone,  i .  o  gram. 

Agar,  i .  5  grams. 

Water,  100.0  c.c. 

Make  the  reaction  about  +2. 

Before  inoculating  media  with  moulds,  some  recommend  placing  the  material  in 
60%  alcohol  for  one  or  two  hours  to  kill  the  bacteria.  The  moulds  withstand  such 
treatment. 

In  cultivating  moulds  it  is  best  to  use  small  Erlenmeyer  flasks,  containing  about 
Y±  in.  of  media  on  the  bottom,  for  the  development  of  the  colonies.  In  order  to 
separate  the  mould  we  may  take  the  hair  or  scales  on  a  sterile  slide  and  cut  them 
into  small  fragments  with  a  sterile  knife.  Then  moisten  a  platinum  loop  from 
the  surface  of  an  agar  slant,  touch  a  fragment  with  the  loop,  and  when  it  adheres 
transfer  it  to  the  agar  slant.  Make  four  or  five  inoculations  on  the  surface  and  from 
suitable  growth,  after  four  to  seven  days,  inoculate  the  medium  in  the  Erlenmeyer 
flask.  Esmarch  roll  cultures  are  better  than  flask  ones. 

Plauth  recommends  receiving  the  mould  material  between  two  sterile  glass  slides. 
Seal  the  edge  of  the  slide  with  wax  and  place  the  preparation  in  a  moist  chamber  for 
four  to  seven  days.  From  developing  fungus  growth  inoculate  the  medium  in  the 
Erlenmeyer  flask.  A  Petri  dish  containing  several  layers  of  thoroughly  moistened 
filter-paper  in  top  and  bottom  makes  a  satisfactory  moist  chamber. 

For  the  study  of  the  morphology  of  Monilia  in  cultures,  Boggs  used 
stab  cultures  of  15%  gelatin.  The  growth  in  the  tube  was  hardened 
in  10%  formalin,  the  glass  cracked  off  and  sections  of  the  gelatin 
column  cut  across  at  any  desired  level.  These  blocks  were  sectioned 
with  the  freezing  microtome;  stained  in  dilute  aqueous  fuchsin  (i  to 
30)  for  several  hours;  then  differentiated  in  saturated  solution  of  citric 
acid  until  nearly  decolorized.  The  sections  were  floated  on  slides,  air 
dried  without  blotting,  cleared  in  xylol  and  mounted  in  balsam. 

For  staining  fungi  in  sections  of  tissue  Busse  recommends  the  folio  wing  method: 
i.  Haematoxylin,  10  to  15  minutes,  then  wash  in  tap  water.  2.  Carbol  fuchsin 
(i  to  20)  30  minutes  or  over  night.  Decolorize  in  alcohol  for  a  few  minutes 
then  through  absolute  alcohol  and  xylol  to  mount  in  balsam.  The  moulds  are  red. 


CHAPTER  XI 
BACTERIOLOGY  OF  WATER,  AIR,  MILK,  ETC. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER 

WHILE  in  a  chemical  examination  as  to  the  character  of  a  water 
there  are  certain  relations  between  the  free  and  albuminoid  ammonias, 
nitrates,  nitrites,  chlorides,  etc.,  which  indicate  the  probable  animal  as 
against  vegetable  nature  of  the  organic  matter  present,  yet  it  is  a  more 
or  less  presumptive  evidence.  In  a  bacteriological  examination  of 
water  the  finding  of  the  colon  bacillus  may  from  a  practical  standpoint 
be  considered  as  positive  evidence  of  human  faecal  contamination. 
Theoretically,  the  possibility  of  organisms  being  present  corresponding 
culturally  to  B.  coli  and  derived  from  cereals  is  to  be  considered.  Also 
the  faeces  of  animals  contain  an  organism  which  cannot  be  differentiated 
from  the  colon  bacillus. 

In  detecting  sewage  contamination  in  water  to  which  varying  amounts  of  sewage 
had  been  added,  it  was  found  that  the  bacterial  tests  were  from  10  to  100  times 
more  delicate  than  the  chemical  ones. 

As  showing  sewage  contamination  of  water,  the  presence  of  the  B.  coli  has  been 
generally  accepted  as  the  most  satisfactory  indication.  The  English  authorities 
consider  sewage  streptococci  and  the  spore-bearing  B.  enter itidis  sporo genes  as  of 
value  as  indicators  as  well  as  the  B.  coli — the  presence  of  sewage  streptococci  indicat- 
ing very  recent  sewage  contamination  and  that  of  the  B.  enteritidis  spcro genes,  in  the 
absence  of  streptococci  and  colon  bacilli,  as  evidence  of  sewage  contamination  at  some 
period  more  or  less  remote. 

In  the  United  States  the  colon  bacillus  alone  is  considered  the  indi- 
cator of  sewage  contamination,  and  all  tests,  presumptive  or  positive, 
are  based  on  the  presence  of  this  organism. 

It  is  not  the  finding  of  the  colon  bacillus  but  rather  the  question  of 
its  relative  abundance  that  is  involved  in  a  water  analysis.  Thus  the 
finding  of  one  colon  bacillus  in  50  c.c.  of  water  would  not  have  weight  as 
showing  contamination,  but  the  presence  rather  constantly  of  the  colon 
bacillus  in  i  c.c.  or  less  makes  contamination  of  a  water  supply  probable. 

In  collecting  samples  of  water  for  bacteriological  examination,  the  following  points 
should  be  considered: 

149 


150  BACTERIOLOGY   OF   WATER,   AIR,   MILK,   ETC. 

i.  The  bottles,  which  should  have  a  capacity  of  from  25  to  100  c.c.,  should  be 
sterile.  Sterilization  may  be  effected  by  heat  or  by  rinsing  with  a  little  sulphuric 
acid  and  subsequently  washing  out  thoroughly  with  the  suspected  water  before  col- 
lection. The  utmost  care  must  be  exercised  that  the  fingers  do  not  come  in  contact 
with  the  glass  stopper  of  the  neck  of  the  bottle  while  filling  it.  If  the  specimen  is 
to  be  sent  some  distance,  it  should  be  packed  in  ice  to  prevent  bacterial  development. 
Frankland  states  that  a  count  of  1000  became  6000  in  six  hours  and  48,000  in  forty- 
eight  hours.  In  water  packed  in  ice  for  a  considerable  time,  however,  the  bacterial 
count  may  diminish. 

2.  If  collecting  from  city  water  supplies,  secure  the  sample  direct  from  the  mains 
and  let  the  water  run  from  the  tap  a.few  minutes  before  collection.  If  the  water  be 
taken  from  a  pond,  stream,  or  cistern,  be  sure  that  the  specimen  comes  from  at  least 
10  inches  below  the  surface.  As  sedimentation  is  the  most  important  method  in 
self-purification  of  rivers  and  ponds,  it  will  be  understood  that  any  stirring  up  of  the 
mud  on  the  bottom  will  enormously  increase  a  bacterial  count. 


Quantitative  Bacteriological  Examination 

i.  Deliver  definite  quantities  of  the  water  to  be  examined  into  tubes  of  liquefied 
gelatin  or  agar  and  plate  out  the  same  in  a  series  of  Petri  dishes. 

A  more  practical  method  is  to  deliver  the  water  from  the  graduated  pipette  into 
the  empty  sterile  dish.  The  water  should  be  deposited  in  the  center  of  the  plate  and 
the  melted  gelatin  or  agar  poured  directly  on  the  water  and  then,  carefully  tilting  to 
and  fro,  mix  the  water  and  the  media.  One  set  of  plates  should  be  of  gelatin  and 
incubated  at  room  temperature;  a  similar  set  should  be  of  lactose  litmus  agar  and 
incubated  at  38°C.  If  the  water  is  highly  contaminated,  it  is  necessary  to  dilute 
it;  thus,  with  river  water,  which  may  contain  from  2000  to  10,000  bacteria  per  c.c., 
a  dilution  of  i  to  100  would  be  desirable. 

Ordinarily  it  will  be  sufficient  to  deliver  from  a  sterile  graduated  pipette  0.2,  0.3, 
and  0.5  c.c.  of  the  water  in  each  of  two  sets  of  plates:  one  set  for  gelatin,  the  other 
for  agar. 

When  gelatin  is  not  at  hand  or  convenient  to  work  with,  the  gelatin  plates  may 
be  replaced  by  others  of  lactose  litmus  agar  for  incubation  at  room  temperature. 
After  twenty-four  hours  at  38°C.  or  forty-eight  hours  at  2o°C.,  the  count  should  be 
made. 

Example. — Forty  colonies  were  counted  on  the  gelatin  plate  containing  0.2  c.c. 
(^5)  of  the  water.  The  number  of  organisms  would  be  200  per  c.c.  Ten  colonies 
were  counted  on  the  agar  plate  containing  0.2  c.c.  and  incubated  at  38°C.  Number 
of  bacteria  developing  at  body  temperature  equals  50  per  c.c. 

There  is  no  strict  standard  as  to  the  number  of  bacteria  a  water  should  contain  per 
c.c.  Koch's  standard  of  100  colonies  per  c.c.  is  generally  given.  It  is  by  the 
qualitative  rather  than  the  quantitative  analysis  that  one  should  judge  a  water. 

If  there  should  be  very  many  colonies  on  a  plate,  the  surface  can  be  marked  off 
into  segments  with  a -blue  pencil.  If  very  numerous,  cut  out  of  a  piece  of  paper  a 
space  equal  to  i  sq.  cm.  By  counting  the  number  of  colonies  inclosed  in  this 
space  at  different  parts  of  the  plate,  we  can  strike  an  average  for  each  space  of  i 
sq.  cm.  To  find  the  number  of  such  spaces  contained  in  the  plate,  multiply  the 


BACTERIOLOGY   OF  WATER  151 

square  of  the  radius  of  the  plate  by  3.1416.  Then  multiply  this  number  by  the 
average  per  square  centimeter,  and  we  have  the  total  number  of  colonies  on  the 
plate.  This  is  the  principle  of  the  Jeffers  disc. 

The  relative  proportion  between  the  bacterial  count  at  2o°C.  and  that  at  38°C. 
is  of  great  importance  from  a  qualitative  standpoint,  as  will  be  seen  later. 

2.  Deliver  into  a  series  of  Durham  fermentation  tubes  containing  glucose  bouillon 
and  into  another  series  containing  lactose  bouillon  varying  definite  amounts  of  the 
water  to  be  examined.  In  tubes  showing  the  presence  of  gas  in  both  glucose  and 
lactose  bouillon  the  evidence  is  presumptive  that  the  colon  bacillus  is  present.  For 
the  positive  demonstration  plates  must  be  made  from  such  tubes  as  show  gas. 

It  is  sufficient  to  deliver  from  graduated  pipettes  in  each  series  quantities  of 
water  varying  in  amount  from  o.i  c.c.  to  10  c.c.  In  our  laboratory  we  inoculate 
with  o.i  c.c.,  0.2  c.c.,  0.5  c.c.,  i  c.c.  and  10  c.c.  of  the  suspected  water.  If  the  o.i  c.c. 
tubes  show  gas,  we  have  reason  to  assume  that  the  water  contained  at  least  10  colon 
bacilli  per  c.c.  If  only  the  10  c.c.  tubes  showed  gas — those  with  less  amounts  not 
having  gas — we  would  be  in  a  position  to  state  that  the  water  contained  the  colon 
bacillus  in  quantities  of  10  c.c.,  but  not  in  quantities  of  i  c.c.  or  less.  Many  authori- 
ties regard  water  as  suspicious  only  when  the  colon  bacillus  is  present  in  quantities 
of  10  c.c.  or  less;  waters  of  good  quality  frequently  showing  the  presence  of  the  colon 
bacillus  in  quantities  of  100  to  500  c.c. 

It  is  generally  accepted  that  if  a  water  shows  the  presence  of  the 
colon  bacillus  in  quantities  of  i  c.c.  or  less,  it  should  be  regarded  as 
suspicious. 

At  the  present  time  the  medium  that  gives  the  least  source  of  error  in  carrying 
out  the  quantitative  presumptive  tests  is  the  lactose  bile.  It  is  made  by  adding 
i  %  of  the  lactose  and  i  %  of  peptone  to  ox  bile,  and  fermentation  tubes  of  the  media 
showing  gas  may  be  considered  as  very  probably  containing  the  colon  bacillus.  The 
percentage  of  error  with  this  method  is  reported  to  be  only  11%,  while  with  glucose 
fermentation  tubes  the  error  is  more  than  50%.  Gas  formation  is  usually  shown  in 
forty-eight  hours,  but  it  is  advisable  to  continue  the  incubation  for  seventy-two  hours. 
These  presumptive  tests  are  chiefly  of  value  in  highly  contaminated  waters.  Even 
with  this  method  plates  should  be  made. 

3.  As  the  colon  and  sewage  streptococci  ferment  lactose  with  the  production  of  acid 
and  hence  produce  pink  colonies  on  lactose  litmus  agar,  much  information  can  be 
obtained  from  the  proportion  existing  between  the  number  of  pink  colonies  and 
those  not  having  such  a  color.  Waters  of  fair  degree  of  purity  rarely  give  any  pink 
colonies. 

Qualitative  Bacteriological  Examination 

General  Considerations.— In  some  countries  the  proportion  of 
liquefying  to  nonliquefying  colonies  on  gelatin  plates  is  considered  of 
importance.  Certain  sewage  organisms  belonging  to  the  proteus  and 
cloaca  groups  liquefy  gelatin;  consequently,  if  the  proportion  of  liquefy- 
ing to  nonliquefying  be  greater  than  as  i  to  10,  the  water  is  considered 


152  BACTERIOLOGY    OF   WATER,    AIR,    MILK,    ETC. 

suspicious.     The  test  is  not  considered  by  American  authorities  as  of 
any  particular  value. 

The  American  Public  Health  Association  recognizes  the  importance  of  the  informa- 
tion obtained  from  a  comparison  of  the  number  of  organisms  developing  at  38°C. 
and  those  developing  at  20°C.  Bacteria  whose  normal  habitat  is  the  intestinal  canal 
naturally  develop  well  at  body  temperature,  while  normal  water  bacteria  prefer  the 
average  temperature  of  the  water  in  rivers  and  lakes.  Consequently  when  the 
number  of  organisms  developing  at  38°C.  at  all  approximates  the  number  developing 
at  2o°C.,  there  is  a  strong  suspicion  that  sewage  organisms  may  be  present.  Normal 
waters  give  proportions  of  i  to  25  or  i  to  50,  while  in  sewage  contaminated  waters  the 
proportion  may  be  as  i  to  4  or  less. 

In  addition,  the  appearance  of  pink  colonies  on  the  lactose  litmus 
agar  is  of  great  assistance  in  judging  of  a  water.  Both  sewage  strepto- 
cocci and  the  colon  bacillus  give  pink  colonies — those  of  the  streptococci 
are  smaller  and  more  vermilion  in  color.  Microscopic  examination 
will  differentiate  the  cocci  from  the  bacilli.  It  is  well  to  bear  in  mind 
that  the  pink  colonies  after  twenty-four  hours  may  turn  blue  in  forty- 
eight  hours  from  the  development  of  ammonia  and  amines.  Conse- 
quently the  lactose  litmus  agar  plates  should  be  studied  after  twenty- 
four  hours. 

A  good  water  supply  will  rarely  show  a  pink  colony,  while  in  a  sewage-contami- 
nated one  the  pink  colonies  will  probably  predominate. 

A  commission  composed  of  eminent  American  bacteriologists  and 
sanitarians  have  recommended  the  following  as  maximum  limits  of 
bacteriological  impurity : 

1.  The  total  number  of  bacteria  developing  on  standard  agar  plates, 
incubated  twenty-four  hours  at  37°C.;  shall  not  exceed  100  per  c.c. 
Provided  that  the  estimate  shall  be  made  from  not  less  than  two  plates, 
showing  such  numbers  and  distribution  of  colonies  as  to  indicate  that 
the  estimate  is  reliable  and  accurate. 

2.  Not  more  than  one  out  of  five  10  c.c.  portions  of  any  sample 
examined  shall  show  the  presence  of  organisms  of  the  Bacillus  coli 
group  when  tested  as  follows: 

(a)  Five  10  c.c.  portions  of  each  sample  tested  shall  be  planted,  each  in  a  fermenta- 
tion tube  containing  not  less  than  30  c.c.  of  lactose  peptone  broth.  These  shall  be 
incubated  forty-eight  hours  at  37°C.  and  observed  to  note  gas  formation. 

(6)  From  each  tube  showing  gas,  more  than  5%  of  the  closed  arm  of  fermenta- 
tion tube,  plates  shall  be  made,  after  forty-eight  hours'  incubation,  upon  lactose 
litmus  agar  or  Endo's  medium. 

(c)  When  plate  colonies  resembling  B.  coli  develop  upon  either  of  these  plate 


THE  COLON  GROUP  153 

media  within  twenty-four  hours,  a  well-isolated  characteristic  colony  shall  be 
fished  and  transplanted  into  a  lactose-broth  fermentation  tube,  which  shall  be 
incubated  at  37°C.  for  forty-eight  hours. 

For  the  purpose  of  enforcing  any  regulations  which  may  be  based  upon  these 
recommendations  the  following  may  be  considered  sufficient  evidence  of  the  presence 
of  organisms  of  the  Bacillus  coli  group. 

F*V^.— Formation  of  gas  in  fermentation  tube  containing  original  sample  of  water. 

Second. — Development  of  acid-forming  colonies  on  lactose  litmus  agar  plates  or 
bright  red  colonies  on  Endo's  medium  plates,  when  plates  are  prepared  as  directed 
above  under  (6). 

Third. — The  formation  of  gas,  occupying  10%  or  more  of  closed  arm  of  the 
fermentation  tube,  in  lactose  peptone  broth  fermentation  tube,  inoculated  with 
colony  fished  from  twenty-four-hour  lactose  litmus  agar  or  Endo's  medium  plate. 

These  steps  are  selected  with  reference  to  demonstrating  the  presence  in  the  sample 
examined  of  aerobic  lactose-fermentating  organisms. 

3.  It  is  recommended,  as  a  routine  procedure,  that  in  addition  to 
five  10  c.c.  portions,  one  i  c.c.  portion,  and  one  o.i  c.c.  portion  of  each 
sample  examined  be  planted  in  a  lactose  peptone  broth  fermentation 
tube,  in  order  to  demonstrate  more  fully  the  extent  of  pollution  in 
grossly  polluted  samples. 

The  members  of  the  B.  coli  group  are  separated  by  the  American 
Public  Health  Association  according  to  fermentation  activities  as 

follows : 

B.  Coli  Group. 
Dextrose  + 
Lactose  + 


Dulcite  +  Dulcite  — 

B.  communior  B.  aerogenes 

B.  communis  B.  acidi-lacti 


i  i 

Saccharose  +  Saccharose  -  Saccharose  +  Saccharose  - 

B.  communior  B.  communis      B.  aerogenes  B.  acidi-lactici. 

Further  divisions  are  made  by  the  use  of  mannite  and  raffinose,  giving  varieties  of 
the  above  four  species. 

Dulcite,  like  mannite,  is  a  hexatomic  alcohol.  It  is  isomeric  with  mannite  and  is 
present  in  Madagascar  manna. 

Raffinose  is  a  trisaccharide;  maltose,  lactose  and  saccharose  are  disaccharides,  while 
dextrose  and  l<evulose  (hexoses)  arabinose  and  xylose  (pentoses)  are  monosaccharides. 

The  diagnostic  characteristics  considered  important  by  the  Ameri- 
can authorities  in  reporting  the  colon  bacillus  (recently  designated 
excretal  colon  bacillus)  are: 


154  BACTERIOLOGY   OF   WATER,   AIR,   MILK,   ETC. 

1.  Typical  morphology,  nonsporing  bacillus,  relatively  small  and  often  quite  thick. 

2.  Motility  in  young  broth  cultures.     (This  is  at  times  unsatisfactory,  as  some 
strains  of  the  colon  bacillus  do  not  show  it  even  in  young  bouillon  cultures). 

3.  Gas  formula  in  dextrose  broth.     Of  about  50%  of  gas  produced,  one-third 
should  be  absorbed  by  a  2%  solution  of  sodium  hydrate  (CO2).     The  remaining  gas 
is  hydrogen.     (Later  views  indicate  that  the  gas  formula  is  exceedingly  variable  and 
should  not  be  depended  upon.     To  carry  out  this  test  one  fills  the  bulb  of  a  fermenta- 
tion tube  with  the  caustic  soda  solution,  holding  the  thumb  over  the  opening  or  with 
a  rubber  stopper,  the  bouillon  culture  and  the  soda  solution  are  mixed  by  tilting  the 
fermentation  tube  to  and  fro.     The  total  amount  of  gas  is  first  recorded  and  then 
that  remaining  after  the  CO2  has  been  absorbed  is  reported  as  hydrogen.) 

4.  Nonliquefaction  of  gelatin. 

5.  Fermentation  of  lactose  with  gas  production. 

6.  Indol  production. 

7.  Reduction  of  nitrates  to  nitrites. 

To  these  may  be  added  the  acidifying  and  coagulation  of  litmus  milk  without 
subsequent  digestion  of  the  casein.  The  production  of  gas  and  fluorescence  in  glu- 
cose neutral  red  bouillon  is  also  a  very  constant  function  of  the  colon  bacillus.  B. 
coli  aerogenes  is  similar  to  B.  coli  with  the  exception  of  nonmotility,  formation  of  gas 
in  starch  media  (bubbles  on  potato  slant)  and  frequent  lack  of  indol  production. 
It  is  often,  especially  in  milk  cultures,  provided  with  a  capsule  and  rarely  forms 
chains.  It  is  a  member  of  the  Friedlander  group  but  differs  from  the  typical  pneu- 
mobacillus  by  producing  acid  and  gas  in  lactose  broth  and  by  its  coagulation  of 
milk. 

B.  coli  anaerogenes  is  also  similar  to  B.  coli  but  does  not  produce  gas  in  glucose  and 
lactose.  This  latter  organism  is  not  usually  recognized  by  American  authorities 
but  I  have  found  on  Endo  plates  an  organism  showing  the  red  colony  with  metallic 
luster  which  failed  to  produce  gas  in  either  glucose  or  lactose. 

NOTE.  —  The  reduction  of  neutral  red  with  a  greenish-yellow  fluorescence  is  very 
striking  and  has  been  suggested  as  a  test  for  the  colon  bacillus.  Many  other  organ- 
isms, especially  those  of  the  hog  cholera  group,  have  this  power.  It  is  convenient, 
however,  to  color  glucose  bouillon  with  about  i%  of  a  %%  solution  of  neutral  red. 


On  the  plates  made  for  the  detection  of  colon  bacillus  may  be  found 
certain  organisms  which  have  origin  in  fecal  contamination.  The  more 
important  of  these  are  those  of  the  paratyphoid,  cloaca  and  proteus 
groups.  In  addition,  the  B.  fecalis  alkdigines  has  not  rarely  been 
isolated.  Among  natural  water  bacteria  there  may  be  present  either 
the  liquefying  or  the  nonliquefying  B.  fluorescens.  These  colonies 
have  a  yellowish-green  fluorescence. 

Certain  chromogenic  cocci  and  bacilli  are  found  in  uncontaminated  waters  as 
B.  indicus  or  B.  violaceus.  From  surface  washings  we  obtain  certain  soil  bacteria  as 
B.  mycoides,  B.  subtilis,  B.  megatherium.  One  of  the  higher  bacteria  which  shows 
long  threads,  Cladothrix  dichotoma,  is  common,  and  is  characterized  by  a  brown  halo 
around  its  gelatin  plate  colony. 


BACTERIOLOGY  OF  MILK  155 

Isolation  of  the  Typhoid  Bacillus  from  Water 

This  is  probably  the  most  discouraging  procedure  which  can  be 
taken  up  in  a  laboratory.  Only  the  most  recent  reports  of  such  isola- 
tion from  water  supplies,  which  have  been  verified  by  immunity  reac- 
tions, can  be  accepted  and  of  these  the  number  of  instances  is  exceed- 
ingly small.  Owing  to  the  long  period  of  incubation,  the  typhoid 
organisms  may  have  died  out  before  the  outbreak  of  an  epidemic 
suggests  the  examination  of  the  water  supply. 

There  have  been  various  methods  proposed  for  the  detection  of  the  B.  typhosus  in 
water.  A  method  which  would  offer  about  as  reasonable  a  chance  of  success  as  any 
other  would  be  to  pass  2  or  3  liters  of  the  water  through  a  Berkefeld  filter;  then  to 
take  up  in  a  small  quantity  of  water  all  the  bacteria  held  back  by  the  filter.  Then 
plate  out  on  lactose  litmus  agar  and  examine  colonies  which  do  not  show  any  pink 
coloration.  The  dysentery  bacillus  has  about  the  same  cultural  characteristics  as 
the  typhoid  one,  so  that  it  is  important  to  note  motility.  If  from  such  a  colony 
you  obtain  an  organism  giving  the  cultural  characteristics  of  B.  typhosus,  carry  out 
agglutination  and  preferably  bacteriolytic  tests  as  well.  Some  strains  of  typhoid, 
especially  when  recently  isolated  from  the  body,  do  i\ot  show  agglutination. 

The  Conradi  Drigalski,  the  malachite-green,  and  various  caffeine  containing  plat- 
ing media  have  been  highly  recommended. 

Isolation  of  the  Cholera  Spirillum  from  Water 

The  method  proposed  by  Koch  in  1893  does  not  seem  to  have  been  improved  upon 
by  later  investigators.  To  100  c.c.  of  the  suspected  water  add  i%  of  peptone 
and  i  %  of  salt.  Incubate  at  38°C.,  and  at  intervals  of  eight,  twelve,  and  eighteen 
hours  examine  microscopically  loopfuls  taken  from  the  surface  of  the  liquid  in  the 
flask.  So  soon  as  comma-shape  organisms  are  observed,  plate  out  on  agar.  The 
colonies  showing  morphologically  characteristic  organisms  should  be  tested  as  to 
agglutination  and  bacteriolysis.  Inasmuch  as  the  true  cholera  spirillum  shows  a 
marked  cholera-red  reaction  it  is  well  to  inoculate  a  tube  of  peptone  solution  from 
such  a  colony  and  add  a  drop  of  concentrated  sulphuric  acid  after  incubating  for 
eighteen  hours.  The  rose-pink  coloration  is  given  by  the  cholera  spirillum  with  the 
acid  alone— the  nitroso  factor  in  the  reaction  being  produced  by  the  organism. 

BACTERIOLOGICAL  EXAMINATION  OF  MILK 

A  bacterial  milk  count  is  of  comparatively  little  value  as  showing 
whether  a  milk  is  dangerous  or  not.  As  a  matter  of  fact,  a  milk  which 
contains  several  million  of  bacteria  per  c.c.  might  be  less  dangerous 
than  one  containing  only  a  few  thousand,  especially  if  in  the  latter 
there  were  numerous  liquefiers  and  gas  producers  present.  There Js, 


156  BACTERIOLOGY   OF   WATER,   AIR,   MILK,   ETC. 

however,  one  point  of  importance  in  connection  with  the  quantitative 
estimation  of  bacteria  in  milk,  and  that  is  the  fact  that  in  order  to  keep 
the  development  of  the  bacteria  within  the  limits  of  10,000  to  50,000 
per  c.c.,  it  is  necessary  that  the  requirements  of  cleanliness  in  milking 
and  the  rapid  cooling  of  the  milk  after  obtaining  it  and  the  keeping  of 
the  temperature  below  5o°C.  be  rigidly  observed.  If  a  milk  has  a 
high  count  it  shows  some  error  in  the  handling  of  the  milk.  Anderson 
has  found  that  top  milk  contains  from  10  to  500  times  as  many  bacteria 
as  bottom  milk.  Centrifugally  raised  cream  contains  more  bacteria 
than  that  forming  by  gravity.  In  making  a  quantitative  bacteriolog- 
ical examination,  the  principle  is  the  same  as  with  water. 

Make  a  known  dilution  of  the  milk  with  sterile  water;  add  definite  quantities  of 
this  diluted  milk  to  tubes  of  melted  agar  or  gelatin,  and  pour  into  plates.  The 
diluted  milk  may  also  be  delivered  in  the  center  of  the  plate  and  the  melted  agar  or 
gelatin  poured  directly  on  it,  mixing  thoroughly.  Always  shake  the  bottle  well 
before  taking  sample. 

Example. — Added  i  c.c.  of  milk  to  199  c.c.  of  sterile  water  in  a  large  flask  (500  to 
looo  c.c.).  After  shaking  thoroughly,  take  i  c.c.  of  this  i  :  200  dilution  and  add  it  to 
99  c.c.  of  sterile  water.  Shaking  thoroughly,  we  have  a  dilution  of  i  :  20,000.  Of  this 
we  added  0.5  c.c.  to  a  tube  of  gelatin  or  agar.  After  incubation  the  plate  showed 
75  colonies.  Therefore  the  milk  continued  in  each  c.c.  75  X  2  X  20,000  (dilution) 
=  3,000,000 — the  number  of  bacteria  in  each  c.c.  of  milk. 

Lactose  litmus  gelatin  or  agar  is  to  be  preferred  in  milk-work,  as  the  normal  lactic 
acid  bacteria  produce  reddish  colonies  which  are  very  striking.  A  standard  easily 
attained  for  high-grade,  certified  milk  would  be  5000  to  10,000  per  c.c. 

In  the  qualitative  examination  of  milk,  many  dairies  employ  the  fermentation 
tube,  any  organism  producing  gas  being  considered  undesirable.  Again  liquefying 
organisms,  as  shown  by  the  presence  of  such  bacteria  in  the  gelatin  plates,  are  evidence 
of  probable  contamination  by  faecal  bacteria.  A  question  which  seems  difficult  to 
decide  is  as  to  the  general  nature  of  the  so-called  normal  lactic  acid  bacteria  of  milk. 
Some  describe  them  as  very  short,  broad  bacilli  with  very  small  colonies,  fermenting 
lactose  with  the  formation  of  lactic  acid.  Others  consider  that  the  streptococci  are 
the  organisms  which  are  concerned  with  the  normal  fermentative  changes.  In 
examining  specimens  of  milk  considered  the  best  on  the  market,  1  have  repeatedly 
found  the  small  red  colonies  on  lactose  litmus  agar  to  be  in  chains  of  either  Gram- 
positive  streptococci  or  streptobacilli. 

Acid  Producing  Organisms. — Shippen  considers  the  chief  organism 
concerned  in  the  souring  of  milk  as  B.  giintherii,  but  notes  that  it  is 
the  same  organism  as  S.  lacticus.  All  authors  note  the  difficulty  of 
deciding  whether  the  morphology  is  coccal  or  bacillary.  McGuire 
found  these  organisms  almost  constantly  present  in  the  dung  of  cows. 
The  organisms  he  obtained  from  cow  dung  were  chiefly  members  of 
the  coli-aerogenes  group  and  S.  lacticus. 


BACILLUS  BULGARICUS  157 

Of  the  acid-forming  bacilli  in  milk  we  have  i.  the  B.  lactls  acid  group.  These  are 
oval  cells  about  0.9  microns  by  0.6  microns,  often  in  chains.  They  are  Gram- 
positive  and  nonmotile.  They  may  be  the  same  as  Streptococcus  lacticus  of  Kruse. 
They  curdle  milk  with  a  homogeneous  clot — this  being  due  to  the  fact  that  they  do 
not  produce  gas  in  lactose  media.  2.  The  B.  coli  aerc genes  group.  These  are  gas 
producers.  (See  under  water.)  3.  The  B.  bulgaricus  group.  In  connection  with 
the  organisms  present  in  the  tablets  used  for  treating  milk  to  produce  lactic  acid  for 
the  treatment  of  intestinal  disorders,  and  considered  to  be  normal  lactic  acid  bacteria, 
I  have  found  both  streptococci  and  bacilli.  These  have  all  agreed,  however,  in  not 
producing  gas  in  either  lactose  or  glucose  fermentation  tubes. 

I  have  often  found  the  commercial  fluid  cultures  sterile,  the  great 
acidity  produced  by  B.  bulgaricus  causing  this.  Fresh  tubes  may  show 
an  acidity  of  +12  or  about  10  times  that  of  ordinary  culture  media. 

The  organism  upon  which  special  stress  is  laid  in  these  so-called  lactic  acid  pro- 
ducers is  the  B.  bulgaricus.  This  is  a  large,  nonmotile  organism  with  square  ends 
like  anthrax.  It  often  occurs  in  long  chains  and  does  not  possess  spores.  It  is  Gram- 
positive  and  often  shows  metachromatic  granules  like  those  of  the  diphtheria  bacillus. 
Colonies  show  in  forty-eight  hours  which  resemble  streptococcus  ones,  but  are  more 
contoured  on  the  surface.  Magnified  the  colonies  resemble  young  mould  colonies 
It  grows  better  on  milk  agar  plates  than  on  whey  agar  plates.  The  opacity  of  the 
milk  agar  plate  is  but  a  slight  objection.  It  produces  a  deep  vivid  pink  in  litmus 
milk,  while  milk  streptococci  only  cause  a  light  pink.  It  produces  a  very  large 
amount  of  acid  (3%).  Little  or  no  growth  on  ordinary  laboratory  media  or  bejow 
o°C.  (Op.  temp.  42°C.). 

Heinemann  states  that  it  occurs  normally  in  human  faeces  and  various  fermented 
milks — also  in  gastric  juice  when  HC1  is  absent.  To  isolate,  put  milk  or  faeces  into 
a  broth  containing  0.5%  acetic  acid  and  2%  glucose.  Transfer  to  litmus  milk  after 
twenty-four  hours  and  from  such  tubes  plate  out  on  milk  serum  agar  (coagulate 
boiling  milk  with  a  few  drops  of  acetic  acid,  filter  and  add  i%  peptone,  2%  glucose 
and  1.5%  agar). 

As  they  grow  in  very  acid  media  the  term  acidophilous  is  applied.  It  was  sup- 
posed that  these  bacteria  were  peculiar  to  certain  fermented  milks  as  matzoon  and 
yogurt.  Hastings  has  shown  the  group  to  be  present  in  milk  in  the  United  States 
and  considers  the  source  to  be  the  alimentary  tract  of  cows. 

Milk  Leukocytes. — Another  source  of  information  as  to  the  quality 
of  a  milk  may  be  derived  from  a  study  of  the  number  of  leukocytes  or 
pus  cells  contained  in  i  c.c.  of  the  milk.  It  must  be  understood  that 
cellular  elements  which  differ  only  slightly  from  true  pus  cells  may  be 
found  in  the  milk  of  healthy  cows  and  may  be  found  in  great  numbers. 
Statements  have  been  made  that  such  cells  are  neither  amoeboid  nor 
phagocytic. 

The  Doane-Buckley  method  is  probably  the  most  accurate.  In  this  you  throw 
down  the  cellular  contents  of  10  c.c.  of  milk  in  a  centrifuge  revolving  about  1000 


158  BACTERIOLIGY   OF   WATER,   AIR,   MILK,   ETC. 

times  a  minute  for  ten  to  twenty  minutes.  Then  remove  supernatant  milk  and  add 
0.5  c.c.  of  Toisson's  solution  to  the  sediment.  Instead  of  Toisson's  solution  I  use 
Gram's  iodine  solution  which  brings  out  the  leukocytes  equally  well  and  gives  a  cleaner 
preparation.  You  thus  have  the  leukocytes  of  10  c.c.  contained  in  0.5  c.c.  (Con- 
centrated 20  times.)  Make  a  haematocytometer  preparation  as  for  blood  and 
find  the  average  number  of  cells  for  each  square  millimeter.  Then  multiply  this  by 
10  to  get  the  number  of  cells  in  a  cubic  millimeter.  As  a  cubic  millimeter  is  1000 
times  smaller  than  a  cubic  centimeter,  you  multiply  the  number  per  cubic  milli- 
meter by  1000.  Then,  as  the  milk  was  concentrated  20  times,  you  divide  by  20. 
(If  it  were  diluted  20  times,  you  would  multiply  by  20.) 

Example. — Found  an  average  of  50  cells  per  square  millimeter.  This  would  make 
500  per  cubic  millimeter,  and  500,000  per  c.c.;  then  500,000  divided  by  20  would 
give  25,000. 

There  is  no  agreement  as  to  a  standard  for  allowable  leukocytes.  Even  in  ap- 
parently healthy  animals  they  may  exceed  100,000  per  c.c.  Doane  has  suggested 
500,000  per  c.c.  as  a  preferable  limit. 

The  smear  methods  for  determining  the  number  of  leukocytes  present  do  not 
compare  in  accuracy  with  the  volumetric  ones.  It  is  important,  however,  from  a 
standpoint  of  examining  for  tubercle  bacilli,  etc.,  as  well  as  for  recognition  of  leuko- 
cytes, to  deposit  the  sediment  from  a  centrifuge  tube,  taken  up  with  a  capillary  bulb 
pipette,  on  a  glass  slide.  Smear  out  slightly  and  then  when  dry  fix  with  a  mixture  of 
ether  and  absolute  alcohol.  Flood  with  ether  to  get  rid  of  remaining  fat  and  stain  by 
Gram's  method  or  by  acid-fast  staining. 

To  summarize,  we  may  state  that  the  bacterial  count  is  an  indicator 
of  the  care  used  in  handling  the  milk  while  the  presence  of  harmful 
bacteria  (qualitative  examination)  or  numerous  pus  cells  indicates 
disease  in  the  cow.  During  1912  severe  epidemics  of  sore  throat  due 
to  a  streptococcus,  S.  epidemicus,  were  traced  to  milk  of  cows  having 
probably  suffered  from  mastitis.  In  Baltimore  the  milk  had  been 
pasteurized  by  the  flash  method  which  indicates  the  unreliability  of 
this  process. 

Pasteurization  of  Milk. — The  objections  to  this  method  of  preserving  milk 
have  been  (i)  that  the  lactic  acid  bacteria  which  have  been  by  some  credited  with 
antagonism  to  harmful  bacteria,  would  be  destroyed  by  pasteurization,  (2)  the  more 
rapid  development  of  bacteria  in  milk  that  has  been  pasteurized  (3)  interference  with 
nutritive  qualities  and  (4)  pasteurized  milk  does  not  show  its  deterioration  as  does 
unpasteurized  milk,  thus  failing  to  give  a  clue  as  to  the  age  of  the  milk. 

The  United  States  Bureau  of  Animal  Industry  in  studying  this  important  phase  of 
the  milk  question  has  grouped  the  milk  bacteria  into  three  classes  (a)  acid-forming, 
(b)  putrefactive  (liquefying)  and  (c)  inert  bacteria.  In  their  investigations  it  was 
found  that  many  acid-forming  bacteria  withstood  temperature  as  high  as  i68°F., 
so  that  pasteurized  milk  was  soured  just  as  is  raw  milk,  but  more  slowly.  They  found 
that  pasteurized  milk  showed  fewer  putrefactive  bacteria  than  raw  milk,  so  that 
even  should  it  be  a  fact  that  injurious  toxins  were  produced  by  spore-bearing  putre- 


BACTERIOLOGY  OF  AIR 


159 


factive  organisms  the  development  of  such  organisms  would  be  even  less  in  pasteur- 
ized milk. 

The  statement  so  often  advanced  that  bacteria  develop  more  rapidly  in  pasteurized 
milk  than  in  raw  milk  was  proved  fallacious. 

It  was  recommended  that  holding  the  milk  for  thirty  minutes  at  i45°F.  was  a  far 
better  method  of  pasteurizing  than  quickly  bringing  the  milk  to  a  temperature  of 
i85°F.  (flash  method).  All  admit  the  great  value  of  the  killing  of  important  patho- 
gens (typhoid,  cholera,  streptococci,  etc.). 


BACTERIOLOGICAL  EXAMINATION  or  AIR 

In  Paris  a  cubic  meter  of  air  was  found  to  contain  the  following 
number  of  organisms: 


Suburbs. — Winter, 

Summer, 

City  Hall.— Winter, 
Summer, 


145  moulds, 

245  moulds, 

1345  moulds, 

2500  moulds, 


170  bacteria. 

345  bacteria. 
4305  bacteria. 
9845  bacteria. 


Air  of  hospitals,  especially  after  sweeping,  may  contain  50,000 
bacteria  per  cubic  meter.  There  does  not  seem  to  be  any  particular 
relation  between  the  amount  of  carbon  dioxide  in  air  and  the  bacterial 
content. 

Petri's  Rough  Method. — Exposure  of  a  lactose  litmus  agar  plate  (capacity  100 
sq.  cm.)  for  five  minutes  will  give  the  number  of  organisms  present  in  10  liters  of  air. 
Multiply  by  100  for  i^cu.  m. 


FIG.  44. — Sedgwick -Tucker  aerobiscope.     (Mac  Neat.) 

The  two  groups  of  organisms  usually  found  in  air  are  i.  bacteria  and  2.  moulds. 
Moulds  (spores)  may  be  carried  by  currents  of  air;  bacteria,  however,  are  generally 
carried  about  by  particles  of  dust  or  finely  divided  liquids  (spray).  On  the  lactose 
litmus  agar  plate  staphylococci  and  streptococci  show  as  bright  red  colonies. 

Sedgwick-Tucker  Sterile  Granulated  Sugar  Method. — Sterilize  aerobioscope 
and  introduce  granulated  sugar  on  support.  Again  sterilize  (not  over  i2o°C.  in 
dry-air  sterilizer).  Allow  a  given  quantity  of  air  to  pass  through;  then  shake  the 
sugar  into  wide  part  of  aerobioscope.  Now  pour  in  10  or  15  c.c.  of  melted  gelatin 
(40° C.)  to  dissolve  sugar.  Roll  tubes  as  for  Esmarch  roll  cultures,  and  incubate 
at  room  temperature.  To  draw  air  through  the  aerobioscope,  connect  the  small  end 
with  a  piece  of  rubber  tubing  which  is  attached  to  a  tube  in  the  stopper  of  an  aspirat- 
ing bottle.  Having  poured  a  definite  quantity  of  water  into  the  aspirating  bottle, 


160  BACTERIOLOGY   OF   WATER,   AIR,   MILK,   ETC. 

allow  the  water  to  run  out.  The  same  quantity  of  air  will  be  drawn  through  the 
sugar  of  the  aerobioscope  as  the  amount  of  water  passing  out  of  the  aspirating  bottle. 
The  bacteria  and  moulds  are  caught  by  the  sugar. 

Example. — Passed  10  liters  of  air  through  the  aerobioscope.  The  bacteria  in  this 
quantity  of  air  showed  75  colonies  when  incubated  at  2O°C.  The  unit  being  i 
cu.  m.  or  1000  liters,  we  have  only  obtained  the  bacteria  of  one  hundredth  of  the 
unit.  Hence  multiplying  75  by  100  gives  7500  bacteria  as  present  in  i  cu.  m.  of 
the  air  examined. 

A  very  satisfactory  method  is  to  take  a  test-tube  containing  5  c.c. 
of  sterile  water  and  having  a  rubber  stopper  with  two  perforations, 
one  for  a  long  piece  of  glass  tubing  which  dips  down  into  the  water 
and  a  short  piece  of  glass  tubing  which  is  connected  with  the  aspirating 
bottle  by  rubber  tubing. 

The  air  to  be  examined  is  drawn  through  the  long  tube  and  its  bacterial  or  mould 
content  is  caught  in  the  water.  By  plating  i  c.c.  which  would  represent  one-fifth 
of  the  total  count  for  the  amount  of  air  aspirated  we  can  easily  calculate  the  con- 
tent for  a  cubic  meter. 

In  comparing  the  results  with  the  aerobiscope  with  those  obtainec 
by  exposing  a  plate  as  in  Petri's  method  for  ten  instead  of  five  minutes, 
it  was  found  that  the  latter  was  sufficiently  in  accord  to  make  it  a  satis- 
factory approximate  quantitative  method.  The  simplicity  and  ease  of 
access  to  the  colonies  developing  on  it  make  it  preferable  when  the  air  of 
operating-rooms  or  hospital  wards  is  to  be  examined. 

Of  the  fungi  ordinarily  obtained  in  examinations  of  the  air  the  blue-green  mould 
and 'the  red  yeast  are  the  most  common.  B.  subtilis  and  sarcina  types  of  cocci  are 
the  most  common  bacterial  colonies  found  upon  exposed  plates.  Sewer  air  is  as  a 
rule  free  from  bacteria,  due  probably  to  the  fact  that  bacteria  tend  to  adhere  to 
moist  surfaces.  The  importance  of  Fliigge's  droplet  method  of  contamination  of 
the  air  of  a  room  is  brought  out  in  the  discussion  of  infection  with  pneumonic  plague. 
This  is  an  important  method  in  the  transmission  of  tuberculosis. 


CHAPTER  XII 
PRACTICAL  METHODS  IN  IMMUNITY 

THAT  which  prevents  the  gaining  of  a  foothold  by  disease  organisms 
in  the  animal  body  or  which  neutralizes  their  harmful  products  or  de- 
stroys the  parasites  is  termed  immunity.  In  the  main,  the  question 
of  immunity  hinges  on  the  powers  of  resistance  of  the  human  body 
and  the  aggressiveness  or  virulence  of  the  invading  organism.  It  must 
always  be  kept  in  mind  that  immunity  is  only  relative;  thus  the  fowl, 
which  is  practically  immune  to  tetanus,  may  be  made  to  succumb  by 
reducing  its  resistance  by  refrigeration  or  by  increasing  the  amount  of 
poison  introduced.  The  insusceptibility  which  the  fowl  has  to  tetanus 
or  which  man  has  to  many  diseases  of  animals  is  best  termed  inherent 
or  inborn  immunity,  and  is  at  present  only  a  subject  of  theoretical 
interest.  When  immunity  to  a  given  disease  is  obtained  as  a  result  of 
an  attack  of  the  disease  in  question  or  bvJahoratory  methods  of  inocu- 
lation, this  is  termed  properly  an  acquired  immunity,  and  injthe  former 
case  is  anna.t.ii rally  acquired  immunity  and  in  the  second  an  artificially 
acquired  immunity. 

Immunity  then  may  be  divided  into  that  which  is  inherent  and  that  which 
is  acquired.  Inherent  immunity  is  such  as  is  observed  in  the  resistance  of  Algerian 
sheep  to  anthrax  (ordinary  sheep  are  very  susceptible)  or  the  fowl  to  tetanus  and 
is  of  interest  theoretically  rather  than  practically.  Acquired  immunity  may  be 
brought  about  naturally  as  by  an  attack  of  a  disease  or  artificially  by  laboratory 
measures. 

As  a  result  of  an  attack  of  a  disease,  which  may  be  regarded  as 
accidentally  acquired,  or  in  response  to  the  stimulus  of  the  injection  of 
the  organism  or  its  products,  we  have  developed  in  the  man  so  infected 
or  injected  certain  specific  antagonistic  properties  to  that  organism, 
which  are  usually  demonstrable  in  the  blood-serum  or  other  body  fluids, 
and  to  which  we  apply  the  terms  agglutinating  power,  precipitating 
power,  opsom'c  power,  or  bacteriolytic  power.  The  term  antibody  is 
also  applied.  All  four  powers  may  be  present  together  in  equal  or  in 
varying  degree  or  one  or  more  may  be  absent.  By  agglutinating  power 
we  mean  that  which  causes  evenly  distributed  organisms  to  come 
ii  161 


162 


PRACTICAL   METHODS  IN   IMMUNITY 


together  and  form  clumps.  By  precipitating  power  we  mean  the 
ability  of  such  a  serum  to  cause  precipitates  in  a  clear  bouillon  filtrate 
of  the  specific  bacterium.  Such  antibodies  are  called  precipitins  or 
coagulins.  Bfv^/psonic  power  we  mean  that  antibody  which  so  alters 
tne  resistance  of  bacteria  that  the  phagocytes  ingest  them.  By^pac- 
teriolytic  poj^er  we  mean  that  which  brings  about  disintegration  or  lysis 
of  the  specific  organism.  The  bacterium  which  causes  the  disease  or 
which  is  used  in  inoculation  for  the  production  of  immunity  is  termed 
the  specific  organism. 


FIG.  45. — Receptors  of  the  first  order  uniting" with  toxin.  (Journal  of  the  Ameri- 
can Medical  Association,  1905,  p.  955.)  a,  Cell  receptor;  b,  toxin  molecule;^ c, 
haptophore  of  the  toxin  molecule;  d,  toxophore  of  the  toxin  molecule;  e,  haptophore 
of  the  cell  receptor. 

Artificially  Acquired  Immunity. — Of  the  different  kinds  of  immunity  only  artificial 
immunity  will  be  considered.  This  may  be  obtained  in  two  ways :  i.  By  injecting 
the  bacteria  or  their  products  into  man  or  animals  and  as  the  result  of  the  activity  of 
the  cells  of  the  animal  invaded,  antibodies'  are  formed  which  neutralize  the  toxins 
(anj^tp^is)  or  bring  about  lysis  W  Ine  specific  bacteria  (baclmojysins).  These 
antibodies  which  are  supposed  to  be  thrown  off  (free  receptors)  from  those  body  cells 
which  have  suitable  fixation  powers  for  the  invading  toxin  molecule  or  bacterium 
may  remain  potential  for  months  or  years  and  so  confer  a  more  or  less  enduring 
immunity. 

These  fixation  points  are  known  as  cell  receptors  and  are  intended  for 
the  assimilation  of  various  foodstuffs  by  the  cell.  If  destroyed  by  the 
toxin  or  bacterium  they  are  reproduced  in  great  excess  by  nature. 


ACTIVE  AND  PASSIVE  IMMUNITY 


I63 


Not  only  may  bacteria  act  in  this  way  but  foreign  cells,  such  as  red  cells  or  various 
parenchymatous  cells,  when  injected,  give^rise  to  antagnnisHr  substances  which  act 
as  factors  in  their  destruction— haemolysins  for  red  cells,  cytolysins  jor  different 
parenchymatous  cells.  Such  methods  produce  "  active  immunity." 

The  substance  which  is  injected  and  in  reaction  to  which  antibodies 
are  produced  is  called  a,n]antigen. 

2.  When  we  take  the  serum  of  a  man  or  animal  immunized  actively  and  inject 
it  with  its  contained  antibodies  into  a  second  animal  or  man,  we  confer  an  immunity 
on  the  second  animal;  but  as  his  cells  take  no  active  part  in  the  production  of  the 
immunity,  but  are  only  passive,  we  term  this  immunity  "passive  immunity."  If 
this  serum  which  is  introduced  in  passive  immunity  only  neutralizes  the  toxic  prod- 


FIG.  46. — Receptors  of  the  second  order  and  of  some  substance  uniting  with  one 
of  them.  (Journal  of  the  American  Medical  Association,  1905,  p.  1131.)  c,  Cell 
receptor  of  the  second  order;  d,  toxophore  or  zymophore  group  of  the  receptor;  e, 
haptophore  of  the  receptor;  /,  food  substance  or  product  of  bacterial  disintegration 
uniting  with  the  haptophore  of  the  cell  receptor. 

ucts  of  the  infecting  bacteria,  we  term  it  antitoxic  passive  immunity  and  designate 
the  immune  serum  as  antitoxic  serum.  If  it  destroys  the  organism,  we  call  it_anti- 
microbic  serum,  and  the  immunity,  antimicrobic  passive  immunity.  Some  immune 
sera  are  both  antitoxic  and  antimicrobic. 

Toxins. — It  is  well  to  remember  that  some  organisms  produce  ajsoluble  or^extra- 
cellular  toxin  which  is  given  off  while  the  bacterium  is  alive:  and  in  other  instances 
the  toxin  is  Jntracellular  and  is  only  given  off  when  the  bacterium  disintegrates: 
consequently,  an  antimicrobic  serum  may  cause  the  liberation  of  toxin.  Diphtheria, 
tetanus,  or  b^tujjism  antisera  are  instances  of  antitoxic  sera,  while  practically  all  others 
a7e""antimicrobic.  The  antidysentery  serum  against  Shiga  strains  seems  to  have 
antitoxic  power.  B.  pyocyaneus  also  has  a  soluble  toxin.  There  is  but  one  factor  to 


164 


PRACTICAL   METHODS   IN   IMMUNITY 


consider  in  an  antitoxic  serum  and  that  is  the  protoplasmic  particles  which  are 
thrown  off  from  the  cell  in  response  to  the  injury  incident  to  the  attack  upon  the  cell 
by  the  toxin  particles.  This  free  particle  in  the  circulation  represents  the  entire 
mechanism  of  antitoxic  immunity.  It  is  capable  of  uniting  with  the  toxin  molecule 
and  neutralizing  its  toxic  power,  or  rather  so  binding  its  combining  end  (naptophore 
group)  that  it  is  incapable  of  attaching  itself  to  a  cell,  so  that  the  poisonous  end  of  the 
toxin  (toxophore  group)  cannot  have  access  to  the  cell. 

The  term  toxin,  strictly  speaking,  is  applicable  only  to  such  bacterial 
poisons  as  (i)  require  a  period  of  incubation  before  being  capable  of 
manifesting  toxic  symptoms  and  (2)  can  produce  antitoxins. 

For  further  discussion  of  toxins  and  antitoxins  see  under  diphtheria,  tetanus, 
botulism,  and  pyocyaneus  infections. 


FIG.  47. — Receptor  of  third  order,  and  of  some  substance  uniting  with  one  of 
them.  (Journal  of  the  American  Medical  Association,  1905,  p.  1369.)  c,  Cell  recep- 
tor of  the  third 'order — an  amboceptor;  e,  one  of  the  haptophores  of  the  amboceptor, 
with  which  some  food  substance  or  product  of  bacterial  disintegration  (/)  may  unite; 
g,  the  other  haptophore  of  the  amboceptor  with  which  complement  may  unite; 
k,  complement;  A,  the  haptophore;  z,  the  zymotoxic  group  of  complements. 


Antimicrobic  Sera. — In  antimicrobic  sera  we  have  two  factors  to 
consider,  the  first  is  a  prQt.np1f».smfr  pajtirlp  quite  similar  to  the  anti- 
toxin molecule,  but  which  in  itself  has  no  power  of  injuring  its  specific 
bacterium.  This  particle  is  generally  referred  to  as  the  amboceptor  or 
immune  body.  It  is  the  specific  product  of  the  activity  of  a  specific 
bacterium  or  foreign  cell  against  the  body  cells  attacked.  It  with- 
stands a  temperature  above  q6°C.  and  of  itself  is  incapable  of  injuring 
the  bacterium  in  response  to  whose  attack  it  was  produced.  The 
second  factor  in  the  bacteriolysis  of  the  specific  bacterium,  or  the  haemoly- 


COMPLEMENT  165 

sis  of  the  specific  foreign  cell,  is  something  normally  present  in  the 
serum  of  every  animal,  and  which  is  capable  of  disintegrating  a  foreign 
cell  or  bacterium,  provided  it  can  have  access  to  the  cell  or  bacte- 
rium through  an  intermediary  ambpceptor  (hence  the  amboceptor  is 
sometimes  called  an  intermediary  body).  This  something  is  called 
the  complement.  It  is  by  some  called  alexine,  by  others  cytase 
(Metchnikoff).  ' ^_  _^^^^" 

The  complement  cannot  act  upon  and  destroy  an  invading  bacterium  or  cell  unless 
the  amboceptor  is  present  to  make  the  necessary  connection.  The  complement  is 
destroyed  by  a  temperature  of  s6°C.,  so  that,  if  we  heat  the  serum  from  an  immune 
animal  to  s6°C.,  the  complement  it  naturally  contains  is  destroyed,  and  the  ambo- 
ceptor it  contains,  which  is  not  injured  by  such  a  temperature,  is  incapable  of  de- 
stroying bacteria  or  cells,  unless  we  replace  the  complement  which  has  been  destroyed 
by  fresh  complement.  This  is  done  experimentally  by  adding  the  serum  of  a  non- 
immunized  animal  which  contains  the  complement,  but  no  specifier  immune  body 
(amboceptor),  to  the  heated  serum.  This  is  termed  "activating,"  and  a  serum  so 
treated  is  said  to  be  "activated."  When  an  immune  serum  has  been  heated  to  56°C., 
it  is  said  to  have  been  "inactivated." 

Antirnicrobic  sera  are  not  as  efficient  in  treatment  as  antitoxic  ones. 
It  might  be  that  if  we  could  use  homologous  sera  for  treating  man  instead 
of  the  usual  heterologous  ones  from  the  horse  better  results  might 
obtain. 

It  would  appear  that  a  more  hopeful  outlook  will  obtain  by  combining  serum 
therapy  with  chemo-therapy,  thus  a  combination  of  antipneumococcic  serum  with 
sodium  oleate  seems  capable  of  producing  curative  results  which  neither  alone  can 
bring  about. 

Again,  a  combination  of  vaccination  (active  immunization)  with  the  injection  of 
the  antimicrobic  serum  (passive  immunization)  has  been  thought  by  some  to  be  of 
value. 

When  we  allow  a  mixture  of  bacteria  or  cells  to  remain  in  contact 
with  their  specific  imrrmne^serum  which  has  been  inactivated,  the  ambo- 
ceptors  attach  themselves  to  the  bacteria  or  cells,  so  tnat  now,  upon 
adding  normal  serum  (complement),  these  bacteria  or  cells  are  so 
prepared  or  mordanted  that  the  complement  can  disintegrate  them. 
This  experiment  of  attaching  amboceptors  to  cells  is  termed  sensitizing 
and  cells  so  treated  are  said  to  be  sensitized. 

METHODS  FOR  OBTAINING  IMMUNE  SERA 

While  a  convalescent  from  a  disease  may  be  utilized  to  obtain  an 
antitoxic,  agglutinating,  opsonic,  or  bacteriolytic  serum  against  the 


i66 


PRACTICAL   METHODS   IN   IMMUNITY 


specific  bacterium,  yet  this  is  more  conveniently  obtained  from  an 
animal  which  has  been  immunized  against  the  bacterium  or  cell  in 
question.  The  rabbit  is  the  most  convenient  animal  to  employ  for  the 
production  of  immune  sera  where  the  object  is  to  have  at  hand  a  serum 
for  use  in  diagnosis. 

Where  sera  are  used  on  an  extensive  scale,  as  in  the  production  of 
curative  sera,  larger  animals  are  employed.  There  are  two  applications 
of  serum  diagnosis:  i.  Where  the  bacterium  is  known  and  the  serum 
is  to  be  diagnosed.  2.  Where  the  serum  is  known  and  the  bacterium 
is  to  be  diagnosed. 

The  first  is  employed  by  testing  the  agglutinating  or  bacteriolytic  power  of  the 
serum  taken  from  a  patient  upon  pure  cultures  of  the  organism  which  is  suspected  as 
the  cause  of  the  disease.  The  Widal  test  (agglutination)  is  the  best  instance  of  this 
procedure.  This  method  is  of  practical  value  in  the  diagnosis  only  of  typhoid,  Malta 
fever,  and  para-typhoid.  In  diseases  like  cholera  and  bacillary  dysentery,  the 
disease  has  run  its  course  before  agglutinating  power  becomes  apparent  in  the 
serum.  This  method,  however,  may  be  used  to  prove  that  a  convalescent  has  suf- 
fered from  a  suspected  disease.  Thus,  by  testing  the  agglutinating  power  of  a 
serum,  one  or  two  weeks  after  recovery  from  a  suspicious  case  of  ptomaine  poisoning, 
we  may  be  able  to  demonstrate  that  the  case  in  question  was  cholera.  The  second 
method  has  wider  application,  and  is  the  one  in  which  we  use  the  sera  of  animals 
which  have  been  immunized  with  known  bacteria.  Organisms  isolated  from  urine, 
faeces,  or  blood  of  patients,  or  those  obtained  from  water  or  food  supplies  may  be 
identified  by  testing  the  agglutinating,  opsonic,  or  bacteriolytic  power  of  known 
sera  against  them.  This  has  a  wide  range  of  applicability.  The  testing  of  the 
opsonic  power  of  the  sera  in  man  or  animals  immunized  against  plague,  and  possibly 
cerebrospinal  meningitis,  seems  to  give  more  definite  information  than  do  agglutina- 
tion or  bacteriolytic  tests.  With  the  majority  of  other  organisms,  however,  the 
agglutination  test  is  the  one  almost  always  preferred. 

Even  in  a  small  laboratory  there  are  no  particular  difficulties  in  the  way  of  having 
on  hand  rabbits  immunized  against  typhoid,  paratyphoid,  Malta  fever,  acid- 
producing  and  nonacid-producing  strains  of  dysentery,  cholera,  etc.  Just  as  we 
inject  men  with  vaccines  prepared  from  various  bacteria  in  opsonic  therapy,  so  we 
inject  animals  to  produce  sera  for  diagnosis.  We  may  use  either  a  bouillon  culture 
or  the  growth  on  agar  slants  taken  up  with  salt  solution  as  the  inoculating  material. 
This  is  heated  for  one  hour  at  6o°C.  to  kill  the  bacteria.  Where  we  desire  to  produce 
a  serum  which  will  disintegrate  red  blood  cells  (hiemolytic  serum),  we  inject  intra- 
venously about  i  c.c.  or  intraperitoneally  about  5  c.c.  of  the  washed  red  cells  of  the 
animal  for  which  we  wish  to  produce  a  specific  serum.  For  details  see  method  of 
preparing  haemolytic  amboceptor  serum  under  Noguchi's  modification  of  Wassermann 
test. 

Precipitating  Sera. — For  preparing  a  serum  for  the  biological  blood  test  we  inject 
the  rabbit  intravenously  with  human  serum  in  quantities  of  about  5  c.c.  every  fifth 
day.  About  one  week  after  the  last  injection  the  antiserum  obtained  from  the  in- 
jected rabbit  should  be  strong  enough  for  Ho  c.c.  to  produce  turbidity  when  added 


AGGLUTINATING  SERA 


l67 


to  i  c.c.  of  a  i-iooo  dilution  of  human  serum  in  salt  solution.     Various  controls  are 
necessary  when  used  in  medico-legal  work. 

Agglutinating  Sera. — For  obtaining  an  agglutinating  or  bacteriolytic  serum  for 
bacteria  we  inject  about  i  c.c.  of  the  killed  bacterial  bouillon  culture  subcutaneously 
or  into  the  peritoneal  cavity  of  the  rabbit.  The  easiest  way  to  inject  the  rabbit  is  to 
hold  the  animal  head  down  and  plunge  the  needle  in  the  median  line  into  the  ab- 
dominal cavity,  forcing  in  the  contents  of  the  syringe.  The  intestines  gravitate  down- 
ward and  by  entering  the  needle  below  the  limits  of  the  bladder  we  avoid  injuring 
any  vital  part.  It  may  be  more  satisfactory  to  at  first  inject  only  about  %  c.c.,  and 


FIG.  48.— i,  Red  cells  +  normal  serum.  No  amboceptor.  No  haemolysis.  A, 
Complement;  B,  normal  red  cell.  2,  Red  cells 4-immune 'serum.  Complement  and 
amboceptor.  Haemolysis.  C,  Complement;  D,  amboceptor;  E,  haemolyzed  red 
cell.  3,  Red  cells  +  immune  serum  heated  to  56.0C.  Inactivated.  Complement 
destroyed.  No  haemolysis.  F,  Destroyed  complement;  G,  amboceptor;  H,  red 
cells.  4,  Red  cells  +  heated  immune  serum  +  fresh  serum.  (Activated  by  con- 
tained complement.)  Haemolysis.  I,  Destroyed  complement;  J,  fresh  complement; 
K,  amboceptor;  L,  heemolysed  red  cell.  5,  Diagram  showing  antitoxin  production. 
a,  Toxin  molecule;  b,  antitoxin  molecule;  c,  neutralization  of  toxin  by  antitoxin. 
6,  Diagram  showing  bacteriolysin.  d,  Complement;  e,  amboceptor;  /,  bacillus. 

then  if  there  is  very  little  reaction,  as  shown  by  the  appetite  and  spirits  of  the  rabbit, 
to  inject  about  four  days  later  i  c.c.  About  four  or  five  injections  at  intervals  of 
three  to  five  days  will  usually  produce  an  immune  serum. 

Injection  of  the  antigenic  material  (blood  cells,  serum  or  bacterial  emulsion)  into 
the  marginal  ear  vein  may  be  employed.  With  this  method,  however,  I  have  had 
several  rabbits  die  in  what  was  considered  anaphylactic  shock.  (For  the  method 
of  immunizing  rabbits  to  produce  a  haemolytic  serum  see  Wassermann  test.)  Some 


i68 


PRACTICAL  METHODS   IN   IMMUNITY 


animals  do  not  seem  to  be  capable  of  producing  antibodies,  so  that  it  may  be  neces- 
sary to  use  one  or  more  rabbits  before  a  satisfactory  serum  is  obtained.  The  most 
convenient  way  of  obtaining  serum  for  a  test  is  to  cut  across  one  of  the  marginal 
veins  of  the  rabbit's  ear,  and  collect  the  blood  in  a  Wright's  U-tube.  Centrifugaliz- 
ing,  we  have  the  serum  ready  for  use. 

The  vein  can  be  made  to  stand  out  prominently  by  applying  a  compress  dipped 
into  very  hot  water.  When  a  large  amount  of  serum  is  desired  it  is  better  to  use  a 
test-tube  with  two  pieces  of  glass  tubing  passing  through  a  double  perforated  rub- 
ber stopper.  To  one  of  the  projecting  pieces  of  glass  tubing  a  stout  hypodermic 
needle  is  attached  through  the  medium  of  8  inches  of  rubber  tubing  and  to  the 
second  piece  of  glass  tubing  passing  through  the  stopper  of  the  large  test-tube 
another  piece  of  rubber  tubing  is  attached  for  suction.  To  obtain  blood  from  the 
rabbit  find  the  ensiform  cartilage  and  insert  the  needle  in  the  notch  to  the  left  and 
gently  force  it  upward.  Applying  suction  with  the  mouth  the  blood  flows  into  the 
test-tube  as  soon  as  the  needle  enters  the  heart.  By  placing  the  tube  of  blood  in 
the  refrigerator  the  serum  separates  out  from  the  clot.  The  removal  of  20  to  30  c.c. 
of  blood  does  not  seem  to  affect  the  animals  in  the  least  and  they  can  be  used  in  this 
way  time  and  time  again.  The  immune  body  and  agglutinin  in  serum  remain 
active  for  weeks  when  kept  in  the  refrigerator.  Such  sera  may  also  be  dried  on  paper 
as  for  amboceptor  paper  (Noguchi).  The  complement  and  opsonin,  however,  begin 
to  deteriorate  at  once  and  have  disappeared  by  the  fifth  day.  Consequently,  for 
opsonic  and  bacteriolytic  and  haemolytic  experiments,  fresh  serum — twelve  to  twenty- 
four  hours — must  be  used,  or  it  may  be  activated. 


AGGLUTINATION  TESTS 

There  are  two  methods  of  testing  the  agglutinating  power  of  a 
serum — the  microscopical  and  the  macroscopical  or  sedimentation 
method. 

The  Widal  Reaction. — i.  For  the  microscopical  method  draw  up  serum  to  the 
mark  0.5  of  the  white  pipette.  Then  draw  up  salt  solution  to  the  mark  n.  This 
when  mixed  gives  a  dilution  of  i  to  20.  (It  is  more  convenient  to  make  the  serum 
dilutions  with  a  graduated  rubber  bulb  capillary  pipette.)  One  loopful  of  the  diluted 
serujn  and  one  loopful  of  a  bouillon  culture  or  salt  solution  suspension  of  the  organism 
to  be  testeo" gives  a  dilution  of  i  to  40.  One  loopful  of  the  i  to  20  diluted  serum  and 
3  loopfuls  of  the  bacterial  suspension  give  a  dilution  of  i  to  80.  These  two  dilutions 
answer  in  ordinary  diagnostic  tests.  The  red  pipette  with  a  i  to  100  to  i  to  200 
dilution  may  be  used  where  dilutions  approaching  i  to  1000  are  desired.  Having 
mixed  the  diluted  serum  and  the  bacterial  suspension  on  a  cover-glass,  we  invert  it 
over  a  vaselined  concave  slide  and  examine  with  a  high  power,  a  dry  objective  (}/§ 
i nch) .  It  is  neater  to  press  down  the  vaselined  periphery  of  the  concavity  on  the  cover- 
glass.  This  sticks  to  the  borders  of  the  cover-glass  and  the  preparation  is  easily 
handled.  It  is  simpler  to  make  a  ring  of  vaseline  to  fit  the  cover-glass  and  make 
the  mixture  of  diluted  serum  and  culture  in  the  center  of  this  ring  or  square.  Then 
apply  the  cover-glass,  press  it  down  on  the  vaseline  ring  and  examine  as  with  the 
ordinary  hanging  drop.  In  making  dilutions  it  is  preferable  to  use  salt  solution,  as 


AGGLUTINATION  169 

the  phenomenon  of  agglutination  requires  the  presence  of  salts.  Ordinarily,  thirty 
minutes  is  a  sufficient  time  to  wait  before  reporting  the  absence  of  agglutination. 
Agglutination  is  more  rapid  at  body  temperature  than  at  room  temperature.  In 
reporting  agglutination,  always  give  time  and  dilution.  It  is  absolutely  necessary 
that  a  control  preparation  be  prepared  in  every  instance;  that  is,  one  with  the 
bacterial  culture  alone  or  with  a  normal  serum  of  the  same  dilution  as  the  lowest  used. 
Some  normal  sera  will  agglutinate  in  i  to  10  dilution,  and  group  agglutinations  (as 
paratyphoid  with  typhoid  serum)  may  occur  in  i  to  40  or  possibly  higher.  It  is  very 
unusual  for  sera  to  agglutinate  any  other  bacteria  than  the  specific  one  in  dilutions  as 
high  as  i  to  80. 

Macroscopic  Agglutination. — 2.  For  the  macroscopical  or  sedimentation  test,  take  a 
series  of  small  test-tubes  (%  X  3  inches)  and  deposit  i  c.c.  of  salt  solution  in  each  of 
the  series.  Now,  having  taken  an  empty  test-tube,  drop  4  drops  of  serum  in  it  and 
then  add  1 2  drops  of  salt  solution.  This  approximately  gives  i  c.c.  of  a  i  to  4  dilution 
of  the  serum.  It  is  more  exact  to  make  the  i  to  4  dilution  with  a  graduated  pipette. 
With  a  rubber-bulb  capillary  pipette,  which  has  been  graduated  to  hold  16  drops  or 
i  c.c.,  draw  up  the  contents  of  the  tube  containing  the  i  to  4  serum  and  add  it  to  the 
next  tube  containing  i  c.c.  of  salt  solution.  This  gives  2  c.c.  of  a  dilution  of  i  to  8. 
Now  mix  thoroughly  by  drawing  up  and  forcing  out  with  the  bulb  pipette,  and  then 
withdraw  i  c.c.  and  add  to  the  next  tube  containing  i  c.c.  of  salt  solution.  This 
gives  a  dilution  of  i  to  16.  Having  mixed  as  before,  again  withdraw  i  c.c.  of  the 
mixture  and  add  it  to  the  i  c.c.  in  the  next  tube.  We  now  have  a  dilution  of  i  to  32. 
Again  withdrawing  i  c.c.  and  adding  it  to  the  fourth  tube  containing  i  c.c.  of  salt 
solution  we  have  a  dilution  of  i  to  64.  In  tube  i  there  is  now  i  c.c.  of  a  dilution  of 
the  serum  of  i  to  8;  in  tube  2,  there  is  i  c.c.  of  a  dilution  of  i  to  16;  in  tube  3  of  i  to 
32.  Tube  4  contains  2  c.c.  of  i  to  64.  Now  adding  i  c.c.  of  a  culture  of  typhoid 
or  any  other  organism,  we  have  the  dilution  of  the  serum  in  each  tube  doubled. 
Tube  i  now  contains  a  serum  in  dilution  of  i  to  16,  acting  on  the  bacteria;  tube  2  of 
a  i  to  32;  tube  3  of  a  i  to  64.  Now  place  these  tubes  in  the  incubator  and,  after  two 
to  five  hours  or  overnight,  we  examine  for  the  clearing  up  of  the  supernatant  fluid. 
If  the  serum  in  a  certain  dilution  agglutinates,  the  clumps  gravitate  to  the  bottom 
and  the  upper  part  becomes  clear.  If  so  desired,  these  dilutions  may  be  carried  on 
to  i  to  several  hundred  in  the  same  way.  It  is  safer  to  work  with  dead  cultures  in- 
stead of  living  ones.  To  prepare,  take  a  twenty-four-hour  agar  slant  culture  of 
typhoid  or  paratyphoid  and  emulsify  in  salt  solution  (about  6  c.c.  to  a  slant). 

By  adding  o.i  of  i%  of  formalin  to  the  typhoid  emulsion  and  placing  in  the  ice- 
box the  cultures  will  be  found  sterile  in  about  three  days.  The  emulsion  should  be 
shaken  twice  daily  while  undergoing  sterilization  in  the  ice-box.  Such  cultures 
are  not  easily  contaminated  and  appear  to  retain  their  agglutinable  qualities  for 
several  months.  The  macroscopic  methods  are  preferable  with  such  dead  cultures. 

A  very  convenient  method  in  general  use  in  Germany  is  the  follow- 
ing: Make  dilutions  of  serum  in  ordinary  test-tubes  (%  X  6  inches) 
as  described  for  the  small  test-tubes. 

Then  take  a  loopful  (2  mg.)  of  culture  from  an  eighteen  to  twenty-four-hour-old 
agar  culture  and  emulsify  it  thoroughly  in  the  dilution  in  the  first  test-tube—repeat 
the  process  in  the  second  tube  and  so  on.  This  procedure  is  much  safer  than  when 


I70  PRACTICAL   METHODS   IN   IMMUNITY 

live  cultures  are  added  with  a  pipette.  Again,  the  dilution  is  unchanged  by  this 
addition  whereas  it  is  doubled  when  an  equal  volume  of  culture  is  added  to  the 
diluted  serum.  A  control  should  always  be  made  in  normal  salt  solution.  After  in- 
cubating, observe  flocculent  precipitates  (agglutination)  by  tilting  the  fluid  in  the 
tubes  to  form  a  thin  layer  and  to  obtain  the  most  advantageous  light  and  look  for  a 
fine  curdy  precipitate  (agglutination)  or  a  uniformly  turbid  emulsion  (negative 
reaction). 

The  method  of  using  a  slide  with  two  vaselined  rings,  one  containing 
an  emulsion  in  the  specific  serum  and  the  other  in  salt  solution  is  of 
great  practical  value.  This  method  is  described  under  cholera. 

Pfaundler  under  the  designation  of  a  thread  reaction  showed  that  organisms 
tended  to  grow  in  thread  forms  in  a  culture  medium  containing  the  homologous 
serum.  Mandelbaum  has  suggested  this  as  a  means  of  diagnosing  typhoid.  Take 
ordinary  bouillon  containing  i  %  of  sodium  citrate.  Inoculate  it  with  a  culture  of 
typhoid.  Now  with  a  bulb  capillary  pipette  take  up  i  part  (as  marked  by  a  wax 
pencil)  of  the  patient's  blood  and  15  times  as  much  of  the  citrated  bouillon  just 
inoculated  with  typhoid.  Mix  the  blood  and  citrated  bouillon  on  a  sterile  slide  or  in 
a  test-tube  and  after  drawing  up  into  the  lower  part  of  the  expansion  of  the  capillary 
pipette,  seal  off  the  capillary  end.  Now  place  the  sealed-off  pipette  upright  in  an 
incubator  and  after  four  or  five  hours  take  out  from  the  expanded  end  a  loopf ul  of  the 
clear  supernatant  fluid  (the  blood  cells  settle  to  the  bottom)  and  if  the  typhoid  bacilli 
are  in  chains  instead  of  being  single  and  motile  it  shows  a  positive  reaction. 


PRECIPITIN  REACTIONS 

In  the  diagnosis  of  bacterial  infections  the  agglutinating  tests  are 
so  much  more  satisfactory  that  precipitin  tests  are  rarely  applied. 
As  will  be  noted  under  the  Meningococcus  such  a  test  has  been  recom- 
mended for  the  diagnosis  of  cerebrospinal  fever. 

In  the  technic  of  precipitin  reactions  for  bacteria  one  filters  two  or  three  weeks 
bouillon  cultures  of  a  given  organism  through  a  Berkefeld  filter.  (Precipitinogen.) 
The  filtrate  should  not  only  be  perfectly  transparent  but  also  sterile  as  subsequent 
bacterial  growth  would  give  turbidity  similar  to  a  positive  reaction.  The  precipitin 
containing  serum  is  prepared  by  injecting  rabbits  intravenously  with  bacterial 
filtrates  as  prepared  above  or  with  tne  bacteria  themselves.  The  methods  are  similar 
to  those  for  preparing  agglutinating  or  haemolyzing  sera. 

Test:  To  four  tubes  each  containing  2  c.c.  of  the  bacterial  filtrate 
are  added  increasing  quantities  of  the  serum  to  be  tested;  0.05  c.c.  to  the 
first  tube,  o.i  c.c.  to  the  second  and  0.5  c.c.  to  the  third,  and  i  c.c.  to  the 
fourth.  Controls  of  positive  and  negative  precipitating  sera  should 
also  be  prepared.  The  tubes  are  not  shaken  and  the  reaction  should 
be  allowed  five  or  six  hours  at  room  temperature  before  final  readings 


BIOLOGICAL  BLOOD  TEST  17 1 

are  made.     When,  the  serum  is  strongly  precipitating  the  clouding  of 
the  clear  fluid  should  take  place  in  ten  to  twenty  minutes. 

In  the  biological  blood  lest  rabbits  are  immunized  intravenously  either  with  whole 
blood  taken  in  citrated  salt  or  with  serum  alone.  For  class  work  I  use  the  blood  of  a 
chicken  injected  intravenously  into  a  rabbit.  An  immune  serum  thus  prepared 
contains  haemolysins  as  well  as  precipitins.  The  haemolyzing  effect  of  such  a  serum 
on  the  nucleated  fowl's  red  cells  shows  well  when  examined  in  a  hanging  drop.  The 
bleeding  of  the  rabbit  should  be  done  after  a  period  of  fasting  to  avoid  any  opales- 
cence  of  the  serum. 

Precipitating  sera  should  be  kept  in  the  cold  and  may  have  one-tenth  of  i  %  car- 
bolic acid  added  as  a  preservative,  or  they  may  be  preserved  on  paper  strips. 

The  suspected  blood  stain  should  be  extracted  with  normal  salt  solution  and 
should  be  filtered  until  perfectly  clear.  An  approximate  strength  of  i  to  1000  of  the 
blood  is  desirable.  This  can  be  estimated  as  given  under  albumin  in  urine,  with  the 
U-shape  tubing. 

Test:  Place  2  c.c.  of  the  i  to  1000  extract  of  the  stain  to  be  examined  in  tube  i, 
2  c.c.  of  i  to  5000  in  tube  2,  and  2  c.c.  of  i«to  10,000  in  tube  3,  adding  to  each  tube 
o.i  c.c.  of  the  precipitating  serum. 

In  another  tube  put  2  c.c.  of  a  i  to  1000  salt  solution  dilution  of  the  serum  of  the 
animal  from  which  the  suspected  blood  is  supposed  to  come  and  add  o.i  c.c.  of 
precipitating  serum. 

Various  other  controls  as  with  normal  rabbit  serum,  etc.,  are  necessary  for  medico- 
legal  application. 

The  tubes  should  not  be  shaken  and  may  be  kept  at  room  temperature  or  in  the 
incubator.  A  positive  reaction  appears  in  two  or  three  minutes  as  a  clouding  at  the 
bottom  of  the  tube  which  becomes  a  distinct  precipitate  in  fifteen  or  twenty  minutes. 
Readings  should  be  made  at  the  end  of  twenty  minutes  as  reactions  occurring  sub- 
sequently have  no  significance. 

DEVIATION  OF  THE  COMPLEMENT 

It  has  been  found  that  if  there  is  not  sufficient  immune  body  in  a 
mixture  of  normal  serum,  containing  abundant  complement,  and  bac- 
terial emulsion,  only  a  portion  of  the  bacteria  will  be  destroyed.  In- 
creasing the  amount  of  immune  body  with  a  constant  quantity  of  normal 
serum,  we  reach  a  point  where  all  the  bacteria  are  destroyed.  Now, 
if  we  continue  to  increase  beyond  this  point  the  addition  of  immune 
serum,  the  destruction  of  the  bacteria  ceases,  and  the  cultures  will  again 
contain  myriads  of  living  bacteria  (Neisser-Wechsberg  Phenomenon). 

To  carry  out  the  test,  make  a  series  of  tubes  containing  mixtures  of  bacteria  with 
the  same  quantity  in  each  of  normal  serum.  Thus,  each  tube  contains  %  c.c.  of 
bacterial  emulsion  and  ^  c.c.  of  i  to  10  normal  serum.  Now  inactivate  a  tube  of 
i  to  loo  immune  serum  and  to  each  of  the  tubes  of  normal  serum  and  bacterial  emulsion 


172  PRACTICAL   METHODS  IN  IMMUNITY 

add  increasing  drops  of  the  inactivated  i  to  100  immune  serum.  Thus,  i  drop  to 
No.  i  tube,  2  drops  to  No.  2  tube,  and  so  on.  After  incubating  for  two  hours,  we 
take  a  pipette  and  plate  out  a  fraction  of  a  drop  in  an  agar  plate.  The  limit  at 
which  bacteriolysis  is  complete  is  shown  by  there  being  an  absence  of  colonies. 

Beyond  or  below  that  point  colonies  are  more  or  less  abundant.  The  explanation 
of  this  phenomenon  of  deviation  or  deflection  of  the  complement  is  that  where  we 
have  an  excess  of  amboceptors  for  available  receptors  on  the  bacterial  cells,  only  a 
portion  of  the  amboceptors  can  attach  themselves  to  their  specific  bacteria.  The 
free  amboceptors,  not  being  able  to  form  a  union  with  the  bacterial  cell  receptors 
(for  which  they  have  a  greater  affinity),  combine  with  the  complement  present. 
Unless  the  complement  be  in  excess,  there  will  be  no  free  complement  left  to  join 
on  to  the  amboceptors  attached  to  the  bacterial  cells,  and  consequently  bacteriolysis 
does  not  take  place  and  the  plate  cultures  show  an  abundance  of  colonies. 

Stimson  has  found,  in  titrating  his  complement  and  amboceptor  for  complement- 
fixation  tests,  that  keeping  his  complement  content  constant  and  successively  increas- 
ing the  amount  of  amboceptor  gives  increasing  haemolyzing  effect  up  to  a  certain 
point,  beyond  which  the  further  addition  of  amboceptor  causes  a  lessening  of 
haemolytic  power. 

This  he  regards  as  due  to  deviation  of  complement  and  in  his  tests  he  prefers  to 
keep  a  fixed  amount  of  amboceptor  and  adjust  his  titrations  by  increasing  comple- 
ment rather  than  amboceptor. 

FIXATION  OR  ABSORPTION  OF  THE  COMPLEMENT 

One  of  the  controversies  in  connection  with  the  nature  of  the  com- 
plement is  that  regarding  the  question  of  the  unity  of  complements 
or  whether  there  exist  different  kinds  of  complements  for  different 
amboceptors  (unity  and  multiplicity  of  complement).  To  prove  that 
a  single  complement  will  act  with  varying  amboceptors,  Bordet  and 
Gengou  showed  that  the  same  complement  would  activate  both  haemo- 
lytic and  bacteriolytic  immune  bodies.  If  to  a  mixture  of  typhoid 
bacteria  and  inactivated  typhoid  immune  serum  some  guinea-pig  serum 
is  added  and  the  mixture  allowed  to  remain  at  37°C.  for  two  hours, 
and  then  sensitized  red  cells  be  added  and  the  mixture  again  placed 
in  the  incubator  for  two  hours,  no  haemolysis  will  be  found  to  have 
occurred,  because  the  bacteria  have  absorbed  all  the  guinea-pig  com- 
plement through  the  intervening  typhoid  amboceptors,  and  there  is  no 
complement  left  to  haemolyze  the  red  cells  through  the  specific  red 
blood-cell  amboceptors. 

If,  instead  of  immune  typhoid  serum,  the  serum  of  a  normal  person  had  been  used, 
there  would  have  been  no  amboceptors  to  unite  the  complement  to  the  bacterial 
cells.  The  complement  would  then  be  at  hand  to  unite  with  the  sensitized  red  cells 
subsequently  added  and  bring  about  their  haemolysis,  as  shown  by  the  ruby  red 
color  of  the  fluid. 


REAGINE  AND  ANTIGEN  173 

This  phenomenon  of  Bordet  and  Gengou  has  been  utilized  by  Wasser- 
mann  for  the  diagnosis  of  diseases  where  cultures  are  not  applicable. 
It  is  well  recognized,  however,  that  the  body  in  a  syphilitic  serum 
which  reacts  with  the  antigen  is  not  an  amboceptor  but  a  lipoidophilic 
substance,  which  has  the  property  of  linking  complement  to  the 
lipoidal  antigen.  The  name  reagine  has  been  proposed  for  this  lipoido- 
philic substance.  A  similar  substance  is  present  in  the  serum  of  yaws 
cases  and  the  Wassermann  reaction  is  just  as  constant  in  such  cases  as 
in  syphilis. 

It  is  in  the  diagnosis  of  syphilis  that  it  is  best  known.  It  having,  until  recently, 
been  impossible  to  obtain  cultures  of  Treponema  pallidum,  we  use  an  emulsion  of  the 
liver  of  a  syphilitic  foetus,  which  has  been  filtered  so  as  to  be  clear,  instead  of  a 
culture.  The  syphilitic  liver,  as  can  be  observed  by  staining  according  to  Levaditi's 
method,  is  packed  with  spirochaetes. 

Antigen. — While  Noguchi  has  recently  obtained  pure  cultures  of  the  organism  of 
syphilis  yet  the  antigen  prepared  from  such  cultures  was  not  found  as  satisfactory 
by  Craig  and  Nichols  as  that  from  the  liver  of  a  syphilitic  foetus,  cases  of  syphilis 
which  showed  strongly  positive  tests  with  ordinary  antigen  not  giving  a  positive  test 
with  the  specific  antigen. 

It  has  now  been  found  that  lecithin  or,  preferably,  emulsions  of  various  normal 
organs  may  be  substituted  as  antigen  for  the  syphilitic  liver,  the  antigenic  power 
being  due  to  lipoids.  Aqueous  extracts  contain  in  addition  to  lipoids,  substances  which 
render  the  antigen  unstable — alcoholic  extracts  are  more  stable  and  contain  less 
anticomplement.  The  preparation  of  acetone  insoluble  antigen  is  described  under 
Noguchi's  method  and  that  of  syphilitic  liver  under  the  Wassermann  reaction. 

Many  prefer  to  use  cholesterinized  antigen.     For  its  preparation: 

Prepare  guinea-pig,  beef  or  human  heart  muscle  as  described  under  acetone  in- 
soluble antigen  and  extract  50  grams  of  this  finely  cut  up  muscle  with  500  c.c., 
absolute  alcohol  for  two  weeks  at  37°C.  Then  filter  and  add  to  one-half  this 
filtrate  about  7  grams  of  C.  P.  cholesterin.  Keep  in  37°C.  incubator  over  night  and 
then  keep  at  a  temperature  of  i6°C.  for  three  hours.  This  precipitates  excess  of 
cholesterin.  Filter  and  to  the  filtrate  add  the  other  one-half  of  the  heart  extract, 
giving  an  antigen  half  saturated. 

In  our  laboratory  we  have  found  this  antigen  rather  sensitive  and 
not  altogether  reliable  and  prefer  to  use  the  acetone  insoluble  one  of 
Noguchi. 

For  the  immune  bodies  we  take  the  serum  of  the  patient,  or  if  a  case 
of  locomotor  ataxia  or  general  paresis,  the  cerebrospinal  fluid. 

In  using  cerebrospinal  fluid  it  is  customary  to  employ  o.i  c.c.,  0.2 
c.c.  and  i  c.c.  quantities  instead  of  the  amounts  given  for  blood-serum 
as  directed  in  the  tests  to  follow. 


174  PRACTICAL   METHODS   IN   IMMUNITY 

It  is  usual  to  expect  strong  fixation  with  a  paresis  fluid  in  the  smallest 
amount  noted  above.     For  tabes  use  more  fluid,  as  0.5  and  i  c.c. 

NOGUCHI'S  METHOD 


For  the  suspension  of  red  cells  use  a  J^%  suspension  of  washed 
human  red  cells. 

For  complement  use  fresh  guinea-pig  serum  in  a  dilution  of  i  part 
to  i>£  parts  of  salt  solution  (40%). 

Method.—  Take  four  small  test-tubes  (12  by  125  mm.)  label  la,  ib  and  20,  2&,  re- 
spectively. Into  la  and  ib  each  put  i  drop  of  the  serum  of  the  patient  to  be  tested 
and  into  ia  and  26  each  put  i  equal  size  drop  of  the  serum  of  a  person  known  to  give  a 
positive  test  for  syphilis.  The  small  drop  delivered  by  a  very  finely  drawn-out 
capillary  pipette,  held  vertically,  is  equal  to  about  0.02  c.c.  or  50  drops  to  i  c.c. 
Next  add  to  each  of  all  four  tubes  i  c.c.  of  the  ^  %  suspension  of  washed  red  cells. 
Then  add  to  each  tube  o.i  c.c.  of  the  40%  fresh  guinea-pig  serum.  Now  add 
to  tube  ia  and  tube  2a  each  o.i  c.c.  of  the  i  to  10  antigen  dilution  (opalescent  working 
antigen  emulsion).  Tubes  ib  and  zb  are  controls  not  containing  antigen.  Mix  con- 
tents of  tubes  thoroughly  and  incubate  at  37°C.  for  one  hour  or  for  one-half  hour 
in  a  water-bath.  Now  add  to  each  of  the  four  tubes  2  units  of  the  immune  haemolytic 
serum,  as  measured  off  on  the  amboceptor  paper  strip  —  thus  with  a  paper  of  which  2 
mm.  equals  i  unit,  drop  into  each  tube  4  mm.  of  the  strip. 

The  tubes  without  antigen  (ib  and  2  b)  should  show  good  haemolysis. 
Tube  2d,  that  of  the  known  syphilitic,  with  antigen,  should  not  show 
haemolysis  and  that  of  the  person  examined  (ia)  should  show  haemolysis 
in  case  the  test  is  negative  for  syphilis.  Moderately  positive  cases  may 
show  a  slight  trace  of  haemolysis.  Where  the  tubes  without  antigen 
are  without  colour  (no  haemolysis)  it  shows  that  there  is  some  anti- 
complementary  factor  at  work  and  that  the  tests  should  be  regarded 
as  unsatisfactory. 

This  failure  to  haemolyze  in  the  control  tubes  may  be  due  to  anticomplementary 
substances  in  the  serum  or  to  the  bacterial  contamination  of  the  serum,  the  proteids 
of  the  disintegrating  bacteria  possibly  preventing  haemolysis  by  antigenic  action  and 
thus  absorbing  the  complement,  which  otherwise  would  bring  about  haemolysis. 

It  is  advisable  also  to  employ  the  serum  of  a  person  known  to  be 
free  from  syphilis.  In  this  case  we  should  use  two  additional  tubes, 
30  and  36,  conducting  the  test  as  for  the  syphilitic  control  serum. 

If  a  normal  serum  is  not  used  then  use  a  system  control  in  which  there  are  put  all  the 
reagents  noted  above  except  the  human  sera  from  the  patient  or  the  syphilitic 
control. 

When  examining  a  large  number  of  sera  at  the  same  time  it  is  well  to  use  rubber 


NOGUCHI  TEST  175 

adhesive  plaster  labels  with  the  name  of  the  patient  written  on  it  as  well  as  the  number. 
The  greatest  care  should  be  exercised  to  avoid  mistakes  in  numbers. 

Many  workers  prefer  to  use  inactivated  serum  for  the  test.  In  this  case  we  should 
add  four  times  as  much  of  the  inactivated  serum  as  for  the  unheated  serum  (0.08 
instead  of  0.02). 

Inactivation  not  only  destroys  complement  but  likewise  diminishes 
the  strength  of  the  reagine  content  of  the  serum.  Factors  such  as 
character  of  food  and  general  condition  influence  the  complement 
strength  of  guinea-pig  serum  so  that  it  is  advisable  to  titrate  the  guinea- 
pig  serum.  To  do  this  take  i  c.c.  of  a  J^%  emulsion  of  human  red 
cells  and  drop  in  i  unit  of  amboceptor  paper.  The  amount  of  com- 
plement which  will  entirely  haemolize  the  red  cells  in  one-half  an  hour  in 
water-bath  equals  i  unit  of  complement.  For  the  test  use  2  units  of 
complement. 

Some  workers  prefer  to  use  a  single  unit  of  complement  and  2  units  of  ambo- 
ceptor, thus  doubling  the  amboceptor  unit.  Stimson  has  found,  however,  that 
increasing  amboceptor  content  may  cause  a  deviation  of  Complement,  so  that  he  prefers 
to  use  a  single  unit  of  amboceptor  and  2  units  of  complement.  In  the  original 
Wassermann  technic  the  units  of  both  complement  and  amboceptor  are  doubled. 
In  the  Noguchi  only  the  amboceptor  unit  is  doubled. 

Preparation  of  Acetone  Insoluble  Antigen.— Take  about  50  grams  of  finely  divided 
beef,  dog,  or  rabbit  heart  or  liver  and  triturate  in  a  mortar  to  a  paste.  Pour  on  this 
paste  500  c.c.  of  absolute  alcohol  and  keep  the  mixture  in  a  corked  bottle  in  the  37°C. 
incubator  for  five  to  seven  days  shaking  the  emulsion  four  or  five  times  daily.  (We 
use  beef  heart  and  carefully  pare  away  all  fat  and  fibrous  tissue  and  macerate  the  re- 
maining muscular  tissue  with  absolute  or  96%  alcohol.  Next  filter  through  paper 
and  collect  the  filtrate  in  a  large  shallow  dish  and  hasten  evaporation  with  the  aid 
of  a  current  of  air  from  an  electric  fan  directed  upon  the  surface.  It  is  advisable  to 
cover  the  dish  with  a  single  layer  of  cheese  cloth  to  prevent  access  of  flies,  etc.,  to  the 
contents.  These  insects  may  contaminate  the  solution  with  moulds  which  causes 
disappearance  of  the  lipoids. 

Within  twenty-four  hours  only  a  sticky  residue  should  remain.  This  is  taken  up 
in  about  50  c.c.  of  ether  and  the  turbid  ethereal  solution  kept  over  night  in  the  re- 
frigerator in  a  corked  bottle. 

In  the  morning  there  will  be  found  about  45  c.c.  of  clear  supernatant  fluid  which  is 
decanted  off  and  allowed  to  evaporate  to  about  15  c.c. 

Now  to  this  15  c.c.  add  about  150  c.c.  of  acetone  and  a  precipitate  will  form 
which  collects  at  the  bottom  of  the  measuring  cylinder.  Now  pour  off  the  supernatant 
acetone  and  let  the  sediment  stand  until  it  is  of  a  resinous  consistence.  Now  dis- 
solve 0.3  gram  in  i  c.c.  of  ether  and  then  add  9  c.c.  of  methyl  alcohol.  This  gives  the 
stock  antigen  solution  which  is  crystal  clear. 

In  using  this  antigen  solution  for  the  Emery  or  Noguchi  test  we  dilute  i  c.c.  with 
9  c.c.  of  salt  solution.  This  opalescent,  working,  antigen  emulsion  should  be  made  up 
fresh  on  the  day  of  preparing  the  tests. 


176        .  PRACTICAL   METHODS   IN  IMMUNITY 

About  one-half  of  these  antigens  are  lacking  in  power  to  absorb 
complement  in  the  presence  of  syphilitic  sera.  More  rarely  they  may 
absorb  complement  with  a  nonsyphilitic  serum  (anticomplementary) 
or  they  may  have  a  haemolytic  action.  Consequently  a  new  stock 
antigen  should  be  tested  as  to  its  reliability — 

1.  A  mixture  of  0.4  c.c.  working  antigen  emulsion,  0.6  c.c.  salt  solution,  and  o.i 
c.c.  of  a  10%  suspension  of  washed  red  cells  when  incubated  at  37°C.  for  two  hours 
should  not  show  any  haemolysis. 

2.  A  mixture  of  0.4  c.c.  working  antigen  emulsion,  0.6  c.c.  salt  solution,  o.i  c.c. 
of  a  40%  solution  of  fresh  guinea-pig  serum,  and  2  units  of  amboceptor  and  incubated 
at  37°C.  for  one  hour  should  show  haemolysis  when  we  now  add  o.i  c.c.  of  a  10% 
washed  red-cell  emulsion  and  the  whole  then  again  incubated  for  two  hours  at  37°C. 
(The  antigen  did  not  absorb  complement  in  the  absence  of  syphilitic  antibodies.) 

3.  A  mixture  of  0.2  c.c.  of  a  i  to  10  dilution  of  working  antigen  emulsion,  0.8 
c.c.  of  salt  solution,  i  drop  of  syphilitic  serum,  o.i  c.c.  of  a  40%  dilution  of  fresh 
guinea-pig  serum,  and  2  units  of  amboceptor  should  be  incubated  at  37°C.  for  one 
hour.     When  we  then  add  o.i  c.c.  of  a  10%  suspension  of  washed  red  cells  and 
again  incubate  for  two  hours  we  should  fail  to  obtain  haemolysis.     (The  antigen  can 
absorb  complement  through  the  intermediation  of  syphilitic  antibodies.) 

Preparation  of  Amboceptor  Paper. — In  order  to  secure  blood  from  the  vein  of  a 
man  or  the  heart  of  the  immunized  rabbit,  the  most  convenient  method  is  with  the 
use  of  an  Erlenmeyer  flask  with  a  rubber  stopper  having  two  perforations  in  the 
stopper.  To  one  of  the  projecting  pieces  of  glass  tubing  a  stout  hypodermic  needle 
is  attached  through  the  medium  of  about  8  inches  of  rubber  tubing,  and  the  second 
piece  of  glass  tubing  is  bent  at  an  angle  as  it  leaves  the  stopper  to  provide  a  suction 
tube.  With  a  man,  constrict  the  upper  arm  sufficiently  to  stop  venous  return  with 
an  Esmarck  rubber  bandage  or  a  towel.  Paint  tincture  of  iodine  over  a  prominent 
vein  at  the  bend  of  the  elbow.  Gentle  suction  will  cause  the  blood  to  flow  into  the 
needle  tube  and  thence  into  the  flask  when  the  vein  is  entered. 

The  blood  as  it  is  taken  from  the  arm  should  be  received  in  about  50  c.c.  of  normal 
salt  solution  containing  i%  of  sodium  citrate.  About  28  to  30  c.c.  are  usually  suffi- 
cient. Now  throw  down  this  red-cell  suspension  in  three  or  four  centrifuge  tubes. 
The  resulting  sediment  should  be  washed  and  rewashed  with  salt  solution.  Two 
to  three  washings  with  salt  solution  suffice. 

Now  take  a  large  healthy  rabbit,  shave  the  lower  abdomen  and  paint  the  surface 
with  tincture  of  iodine.  The  easiest  way  to  inject  the  rabbit  is  to  hold  the  animal 
head  down  and  plunge  the  needle  of  a  large  glass  hypodermic  syringe  containing  the 
washed  red-cell  sediment  into  the  abdominal  cavity  in  the  median  line.  The  intes- 
tines gravitate  downward  and  by  entering  the  needle  below  the  limits  of  the  bladder 
we  avoid  injuring  any  vital  part. 

Make  the  injections  at  intervals  of  five  days  and  give  increasing  amounts  at  each 
successive  injection.  Thus,  first  injection,  5  c.c.;  second  injection,  8  c.c.;  third 
injection,  10  c.c.;  fourth  injection  12  c.c.;  and  at  the  fifth  injection  give  about  15  to 
20  c.c.  of  washed  red-cell  sediment.  It  is  well  to  dilute  the  cell  sediment  with  an 
equal  amount  of  salt  solution.  About  ten  days  after  the  last  injection,  we  take  some 
blood  in  a  Wright's  tube  from  a  vein  of  the  ear  and  dilute  the  serum  to  make  a  i  to 


THE  WASSERMANN  TEST  177 


100  dilution.  To  i  c.c.  of  a  J^%  emulsion  of  red  cells  we  add  o.i  c.c.  of  a  40%  dilu- 
tion of  guinea-pig's  fresh  serum  —  similar  combinations  being  made  in  a  series  of 
10  tubes.  To  each  of  these  tubes  we  add  varying  amounts  of  the  i  to  100  dilution, 
o.i  c.c.  in  the  first,  0.2  c.c.  in  the  second,  0.3  c.c.  in  the  third,  and  so  on.  If  we 
obtain  haemolysis  in  the  tube  containing  0.2  c.c.  of  i  to  100  dilution  of  serum  but  not 
in  that  containing  o.i  c.c.  we  note  that  the  serum  has  a  titer  of  about  i  to  500.  If 
the  o.i  c.c.  gave  haemolysis,  the  serum  would  have  a  titer  of  i  to  1000. 

Having  ascertained  that  the  haemolytic  serum  is  sufficiently  strong  we  shave  the 
left  side  of  the  thorax  of  the  rabbit  and  enter  the  needle  of  the  apparatus  similar  to 
that  used  for  taking  the  blood  from  a  man's  vein  in  one  of  the  intercostal  spaces  of  the 
left  side. 

Having  introduced  the  needle,  feel  for  the  heart  beat  and  then  plunge  the  needle 
into  the  heart.  We  can  withdraw  about  30  c.c.  of  blood  without  injury  to  the  rabbit. 
This  blood  should  be  received  in  a  clean  empty  flask  and  set,  over  night,  in  the  re- 
frigerator. The  following  morning  pour  off  the  clear  serum  into  a  clean  Petri  dish 
and  saturate,  one  by  one,  squares  of  filter  paper  with  the  serum.  Allow  the  filter 
paper  to  dry  on  a  piece  of  unbleached  muslin.  Noguchi  recommends  Schleich  and 
Schull's  paper  No.  597.  When  thoroughly  dry  cut  strips  5  mm.  wide.  This  makes 
the  amboceptor  paper.  To  standardize,  take  a  series  of  tubes  containing  i  c.c.  of 
a  K%  emulsion  of  red  cells  and  add  o.i  c.c.  of  40%  dilution  of  guinea-pig  serum  for 
complement.  Next  cut  across  the  amboceptor  paper  strip  pieces  of  varying  width, 
as  i  mm.,  2  mm.,  3  mm.,  5  mm.,  and  so  on.  The  narrowest  strip  which  gives  haemo- 
lysis in  one  hour  in  the  incubator  or  one-half  hour  in  the  water-bath  equals  i  unit. 
Thus  if  a  piece  5  mm.  wide  was  required  to  produce  haemolysis,  5  mm.  of  the  paper 
would  have  a  value  of  i  unit. 

Coca  injects  rabbits  intravenously  with  i  c.c.  of  well-washed  red  cells 
and  five  days  later  gives  a  similar  dose  intravenously.  The  blood  for 
the  haemolytic  serum  is  taken  from  the  rabbit  five  days  after  the  second 
and  last  injection.  He  states  that  such  a  method  not  only  gives  a 
high  titer  but  avoids  to  a  great  extent  agglutination  difficulties.  It 
also  is  more  stable  as  regards  holding  its  original  titer.  We  have  had 
better  success  with  the  following  method  than  with  Coca's.  Inject  in- 
travenously Y±  c.c.  of  50%  red-cell  suspension  and  three  days  later 
inject  ^  c.c.  of  the  same  strength  suspension.  Several  days  later  in- 
ject i  c.c.  and  withdraw  seven  days  afterward. 

THE  WASSERMANN  TEST 

In  the  Wassermann  reaction  the  rabbits  are  injected  with  sheep 
red  cells  which  have  been  washed  twice  with  salt  solution  by  aid  of  the 
centrifuge.  About  five  intraperitoneal  injections  with,  on  the  average, 
the  quantity  of  red  cells  contained  in  5  c.c.  of  sheep  blood,  given  at  in- 
tervals of  five  days,  gives  a  strong  haemolytic  serum  if  taken  about 
one  week  after  the  last  injection. 


12 


178  PRACTICAL   METHODS  IN  IMMUNITY 

The  standardization  of  the  titer  is  similar  to  that  for  the  human  hsemolytic 
amboceptor,  except  that  5%  emulsion  of  sheep  cells  with  0.05  c.c.  undiluted  guinea- 
pig  serum  is  used  instead  of  the  %%  emulsion  of  human  cells  and  the  o.i  c.c.  of  40% 
guinea-pig  serum. 

The  method  is  to  take  in  a  test-tube  0.2  c.c.  inactivated  human 
serum  (heated  for  twenty  minutes  at  56°C.),  o.i  c.c.  fresh  serum  from 
guinea-pig  for  complement,  i  unit  antigen  and  3  c.c.  normal  salt  solu- 
tion; then  to  incubate  for  one  hour  at  37°C.  (An  antigen  unit  is  the 
amount  that  will  inhibit  haemolysis  of  i  c.c.  of  5%  emulsion  of  sheep 
cells  when  mixed  with  0.2  c.c.  luetic  serum  and  o.i  c.c.  guinea-pig 
complement.) 

There  should  likewise  be  absolutely  no  inhibition  with  0.02  c.c.  of  serum  from  a 
normal  individual.  The  unit  is  made  up  to  i  c.c.  with  salt  solution. 

Then  add  2  units  of  amboceptor  and  i  c.c.  5%  emulsion  of  sheep 
red  cells,  shake  and  incubate  for  one  hour.  (The  amount  of  haemolytic 
serum  that  will  haemolize  i  c.c.  of  a  5%  emulsion  of  sheep  red  cells 
to  which  0.05  c.c.  guinea-pig  serum  has  been  added,  in  one  hour,  is  an 
amboceptor  unit.) 

In  our  laboratory  we  use  i  c.c.  of  a  i  to  10  dilution  of  the  clear  stock  acetone 
insoluble  antigen  of  Noguchi  as  the  antigen  unit. 

In  the  preparation  of  the  antigen  originally  recommended  by  Wasser- 
mann,  we  finely  divide  the  liver  of  a  syphilitic  foetus  and  extract  it  for 
twenty-four  hours  with  salt  solution  containing  0.5%  carbolic  acid. 
Frequent  shaking  is  required.  The  supernatant  fluid  is  decanted  and 
centrifuged.  This  turbid  fluid  is  pipetted  off  and  kept  in  the  ice-box  in 
a  brown  bottle  for  several  days.  This  yellowish-brown  opalescent  fluid 
is  the  antigen  and  is  standardized  so  that  i  unit  equals  the  amount 
which  will  inhibit  haemolysis  of  i  c.c.  of  5%  emulsion  of  sheep  red 
cells  when  mixed  with  0.2  c.c.  luetic  serum,  o.i  c.c.  guinea-pig  com- 
plement and  2  units  of  amboceptor. 

The  same  technic  is  employed  with  the  control  test-tube  except  that 
the  antigen  unit  is  not  put  in. 

The  Noguchi  method  has  been  stated  to  give  a  positive  reaction  with  nonsyphilitic 
sera  in  about  7%  of  cases.  The  Wassermann  a  negative  result  in  about  9%  of  syph- 
ilitic sera.  These  figures  show  the  advantage  of  checking  one  against  the  other. 

One  objection  to  the  Wassermann  test  is  that  a  majority  of  human 
sera  show  native  antisheep  amboceptors  and  in  some  instances  the 
amount  of  this  constituent  may  be  so  great,  when  added  to  the  2  units 


EMERY'S  TECHNIC  179 

of  amboceptor  from  the  serum  of  the  sheep  cell  immunized  rabbit,  as 
to  cause  haemolysis  with  a  syphilitic  serum.  Simon  recommends 
treating  the  sera  to  be  tested  with  sheep  cells  in  order  to  fix  these 
native  amboceptors.  Centrif  ugalizing,  the  sheep  cells  with  the  attached 
amboceptors  are  thrown  down,  leaving  the  clear  supernatant  serum 
free  of  these  amboceptors. 

There  seem  to  be  certain  sera  when  with  a  clinical  history  of  syphilis  we  obtain  a 
positive  Wassermann  with  unheated  serum  and  a  negative  one  with  inactivated 
serum.  In  order  to  obtain  information  with  the  same  serum  heated  and  unheated 
I  would  recommend,  when  it  is  inconvenient  to  carry  out  the  original  Wassermann 
technic,  to  employ  the  Noguchi  technic  with  inactivated  serum  and  the  Emery 
technic  with  fresh  unheated  serum.  In  any  case  when  serum  cannot  be  tested  within 
twenty-four  hours  it  should  be  inactivated,  as  unheated  serum  tends  to  become 
anticomplementary. 


EMERY'S  TECHNIC  FOR  THE  WASSERMANN  TEST 

Owing  to  technical  difficulties  with  the  method  of  making  and 
employing  the  antigen  and  amboceptor  features  of  the  original  Emery 
test,  I  have  retained  the  principle  of  the  test  but  substituted  the 
reagents  prepared  in  exact  accordance  with  Noguchi's  directions. 

Briefly  stated,  the  principle  of  Emery's  test  consists  in  the  employment  of  fresh 
human  serum  for  supplying  complement  and  the  primary  incubating  of  the  haemolytic 
system  (human  red  cells  and  rabbit  serum  immune  to  human  red  cells)  at  the  same 
time  as  the  incubation  of  the  antigen  and  serum  but  in  separate  tubes.  Then  in  the 
second  period  of  incubation  to  add  these  "sensitized"  cells  to  the  serum  antigen 
combination. 

In  Noguchi's  method  all  reagents  are  incubated  together. in  the 
first  period  with  the  exception  of  the  amboceptor  paper  (dried  serum 
of  rabbit  immune  to  h  iman  red  cells),  which  is  not  added  until  the 
period  of  incubation  for  complement  binding  is  completed  and  the 
second  incubation  commenced.  Time  is  saved  in  the  Emery  technic, 
inasmuch  as  the  red  cells  are  already  sensitized  by  the  haemolytic 
amboceptor  when  added  to  the  tubes,  and  haemolysis  shows  itself 
almost  immediately  in  tubes  where  the  complement  has  not  been 
absorbed  by  the  antigen  through  syphilitic  antibodies. 

Noguchi  has  called  attention  to  the  fact  that  protein  constituents  of  certain 
aqueous  or  alcoholic  extracts  may  have  the  power  to  fix  complement  through  certain 
intermediaries  existing  in  fresh  serum  which,  however,  does  not  obtain  for  inacti- 
vated sera  (sera  heated  to  s6°C.  for  fifteen  minutes). 


i8o 


PRACTICAL    METHODS    IN   IMMUNITY 


Pure  lipoidal  substances  as  contained  in  Noguchi's  acetone  insoluble  antigen, 
however,  do  not  act  in  this  way. 

Consequently  by  using  such  an  antigen  we  eliminate  the  objection  to  employing 
fresh  human  serum  in  the  test  for  syphilitic  antibodies. 

As  giving  more  uniform  haemolytic  results  and  as  being  more  stable  and  easier 
of  employment,  I  have  made  use  of  Noguchi's  directions  for  taking  up  the  serum  of 
the  rabbits,  immunized  to  human  red  cells  and  his  method  of  standardizing  this 
"amboceptor"  paper.  In  practice,  I  measure  off  the  length  of  paper  correspond- 


CONTROL 


U.  40        I  tobO 
ANTIGEN    ANTIGEN   ANTIGEN   ANTIGEN    ANTI&EN  ANTIGEN 

5 


FIG.  49. — i.  Capillary  pipette  being  graduated  by  drawing  up  i  and  4  drops 
from  a  watch-glass,  (a)  Blue  pencil  mark  of  i  drop  or  i  volume,  (b)  Mark  of 
volume  of  4  drops.  2.  Graduated  centrifuge  tube  containing  sodium  citrate  normal 
salt  solution.  3.  Tube  with  10  amboceptor  units  in  i  c.c.  of  salt  solution.  4.  Mix- 
ture of  i  volume  20%  emulsion  red  cells  and  4  volumes  inactivated  amboceptor 
solution.  5.  Small  glass  tubes  for  Emery  test.  6.  Method  of  transferring  from 
tube  to  tube.  7.  Making  a  Wright  U-tube — the  end  "a"  to  be  used  as  a  capillary 
pipette. 

ing  to  10  Noguchi  units  and  dissolve  the  dried  serum  in  such  paper  in  i  c.c.  of  salt 
solution.  This  make  a  satisfactory  and  uniform  substitute  for  the  sterile  immune 
serum  used  by  Emery. 

Method:  i.  Take  blood  from  the  finger  or  ear  in  a  large  Wright  U  tube  (Y±  inch 
in  diameter).  Place  in  37°C.  incubator  for  fifteen  minutes  (to  increase  yield  of 
serum)  and  then  centrifuge. 

Of  course  wheli  the  Noguchi  test  is  being  made  at  the  same  time  we 
use  the  serum  of  the  blood  taken  from  a  vein  in  the  arm. 


EMERY'S  TECHNIC  FOR  WASSERMANN 


181 


2.  Graduate  a  capillary  pipette  for  i  volume  and  4  volumes. 

There  is  the  greatest  variation  in  the  size  of  a  drop  delivered  by  a  capillary  pipette, 
this  difference  in  size  not  only  occurring  with  varying  diameters  of  the  capillary 
tube  but  with  position  of  tube,  thus  a  tube  with  i  mm.  diameter  and  held  horizon- 
tally will  deliver  about  16  drops  of  serum  to  the  c.c.  If  held  vertically  about  32.  A 
fine  capillary  pipette,  such  as  is  used  by  Noguchi  will  deliver,  when  held  vertically, 
50  drops  to  the  c.c.,  or  0.02  c.c.  to  the  drop.  It  is  therefore  better  to  standardize 
with  known  amounts  delivered  from  a  graduated  pipette,  using  J-^Q  c-c-  as  the  un^ 
for  i  volume. 


FIG  50—  i.  Copper  water  bath  12X12X8  inches,  (a)  Thermometer  to  show 
38°C  (b)  Tubes  containing  antigen  dilutions,  (c)  Tube  containing  hamolytic 
system  incubating  along  with  the  antigen  tubes.  2.  Ordinary  rice  cooker  with  copper 
holder  for  test-tubes. 

3.  Into  each  of  a  series  of  small  test-tubes  put  4  volumes  of  normal  salt  solution. 
(These  tubes  are  most  conveniently  made  by  breaking  off  2^-  to  3-inch  lengths  of 
24-inch  soft  glass  tubing  and  then  fusing  one  end  in  the  flame  to  make  a  small  test- 
tube.) 

Make  a  distinguishing  mark,  e.g.,  X,  on  end  of  tube  with  blue-wax  pencil  and  i 
this  tube  to  hold  control.     Mark  the  other  tubes  I,  II,  III,  and  so  on.     When  differ- 
ent sera  are  to  be  tested  they  may  be  distinguished  by  lines  either  above  or  below, 
or  with  circles;  also  marks  with  red-wax  pencil  may  be  used. 

4.  Make  a  i  to  20  dilution  of  stock  antigen  solution  in  salt  solution. 

To  Tube  I  add  4  volumes  of  i  to  20  antigen,  thus  making  8  volumes  of  i  to  40 


1 82  PRACTICAL   METHODS   IN   IMMUNITY 

antigen  in  Tube  I.  Mix  thoroughly  by  manipulating  bulb  of  pipette.  Then  trans- 
fer 4  volumes  of  the  i  to  40  from  Tube  I  to  Tube  II,  and  so  on  through  the  series. 
When  the  dilution  in  the  last  tube  has  been  made  throw  4  volumes  away. 

The  4  volumes  of  dilution  of  the  antigen  in  the  respective  tubes  will  then  be: 
In  Tube  I,  i  to  40;  in  Tube  II,  i  to  80;  in  III,  i  to  160;  in  IV,  i  to  320,  and  so  on. 

5.  Add  i  volume  of  serum  to  be  tested  to  control  tube  X,  and  to  each  of  the  tubes 

I,  II,  III,  etc.,  in  succession.     (If  the  serum  be  added  to  the  antigen  tubes  before  the 
control  tube,  antigen  might  be  carried  over  to  the  control.) 

NOTE. — If  the  serum  has  been  inactivated  restore  complement  by  adding  i  volume 
of  a  40%  fresh  guinea-pig  serum.  Also  use  2  volumes  of  this  inactivated  human 
serum  instead  of  i. 

6.  Incubate  at  38°C.  for  thirty  minutes.     This  allows  syphilitic  antibody,  if 
present,  to  bind  complement. 

7.  As  soon  as  the  above  mixtures  have  been  made  and  put  in  the  incubator 
prepare  the  "haemolytic  system"  by  adding  i  volume  of  20%  emulsion  of  washed 
human  red  cells  to  4  volumes  of  solution  of  amboceptor  paper  (10  Noguchi  units  of 
amboceptor  paper  dissolved  in  i  c.c.  of  salt  solution.    Thus  of  a  paper  of  which  4  mm. 
was  the  unit  we  should  cut  off  about  40  mm.,  place  in  test-tube  and  extract  the  dried 
serum  with  i  c.c.  of  salt  solution),  and  place  this  haemolytic  system  in  incubator 
alongside  the  tubes  already  there.    To  obtain  the  washed  red  cells  allow  4  to  10 
drops  of  blood  to  drop  into  a  graduated  centrifuge  tube  containing  salt  solution  to 
which  has  been  added  i  %  of  sodium  citrate  to  prevent  coagulation.     After  shaking, 
centrifuge.     Pour  off  supernatant  fluid,  replace  with  salt  solution,  again  shake  and 
centrifuge — this  sediment  of  red  cells  is  to  be  diluted  with  4  volumes  of  salt  solution 
(20%  emulsion).     (Incubation  hastens  sensitization  of  the  red  blood  cells.     Agglu- 
tination of  red  cells  may  occur  with  certain  haemolytic  sera.    The  immunization  of 
the  rabbits  with  small  doses  intravenously  (Coca's  method)  tends  to  prevent  this 
interfering  factor.     However,  frequent  shaking  is  usually  sufficient  to  break  up  the 
agglutinating  masses  of  red  cells). 

8.  At  the  expiration  of  thirty  minutes  from  the  commencement  of  incubation 
for  complement  binding,  add  i  volume  of  haemolytic  system  to  each  of  the  tubes,  I, 

II,  III,  etc.,  in  the  order  of  antigen  dilution. 

9.  Finally,  after  washing  pipette  in  salt  solution,  add  i  volume  of  haemolytic 
system  to  control  in  tube  X.     (If  the  haemolytic  system  should  be  added  to  the  con- 
trol tube  before  the  antigen  tubes,  complement  from  the  control  tube  might  be 
carried  over  to  the  antigen  tubes.) 

Shake  each  tube  thoroughly.  Allow  them  to  incubate  for  a  few  minutes.  Then 
examine  tubes  I,  II,  III,  etc.,  for  haemolysis.  The  control  should,  of  course,  show 
haemolysis.  The  antigen  tubes  should  show  a  white,  supernatant  fluid  over  the  in- 
tact red-cell  sediment  in  the  tubes  with  the  low  dilutions  and  even  in  the  highest 
dilutions,  where  the  serum  is  strongly  positive.  In  a  weakly  positive  serum,  in- 
hibition of  haemolysis  may  only  show  in  the  first  tube  and  haemolysis  show  in  those 
tubes  having  higher  dilutions  of  antigen. 

It  will  be  noted  that  the  reagents  are  made  in  accordance  with 
Noguchi's  directions.  Even  in  those  cases  where  fresh  guinea-pig 
serum  is  employed  to  replace  complement,  absent  from  the  inactivated 


USE  OF  SENSITIZED  CELLS  FOR  NOGTJCHI  TECHNIC  183 

human  serum  tested,  we  employ  the  40%  solution  used  in  Noguchi's 
technic. 

Experiments  have  shown  that  one  volume  of  serum  contains  almost  invariably 
sufficient  complement  to  haemolyze  the  red  cells  present.  As  a  matter  of  fact  in  95% 
of  sera  one-half  this  amount  would  suffice. 

It  will  be  noted  that  the  amboceptor  and  antigen  content  of  the 
i  to  80  tube  is  proportionate  to  that  used  in  the  Noguchi  test. 


NAVAL  MEDICAL  SCHOOL  MODIFICATION  OF  NOGUCHI'S  TECHNIC 

There  is  evidence  for  believing  that  the  heating  of  the  serum  to  be 
examined  not  only  destroys  about  75%  of  its  total  syphilitic  antibody 
content  but  may  also  destroy  thermolabile  substances  which  may  be 
of  importance  in  bringing  about  a  positive  reaction.  At  any  rate  I 
have  had  experience  with  sera  where,  with  an  absolutely  clear  diagnosis 
of  syphilis  followed  by  response  to  therapeutic  measures,  there  has 
been  a  negative  reaction  with  inactivated  serum  but  a  positive  one  with 
unheated  serum. 

For  this  reason  I  would  advise  that  the  Noguchi  method  be  carried 
out  with  inactivated  serum  and  an  Emery  test  also  made  using  un- 
heated serum. 

The  Emery  test  is  also  of  value  as  showing  a  quantitative  relation.  At  the  same 
time  I  am  convinced  that  it  is  more  conservative  to  make  a  diagnosis  of  syphilis  only 
with  inactivated  serum,  reserving  the  utilization  of  findings  with  unheated  sera  for 
effect  of  treatment  on  syphilitics  and  for  such  cases  as  the  symptomatology  and 
history  of  the  case  would  indicate  that  a  positive  reaction  should  be  obtained. 

No  injury  is  conferred  on  a  patient  by  a  negative  Wassermann  be- 
cause in  the  presence  of  other  data  treatment  will  not  be  withheld. 

A  modification  of  the  Noguchi  technic,  along  the  Emery  lines,  which  saves  time 
and  makes  readings  sharper  is  to  employ  sensitized  red  cells.  To  do  this  add  to  the 
drop  of  patients  serum  and  40%  guinea-pig  complement  and  antigen  in  tube  la,  % 
c.c.  of  salt  solution.  Also  to  tube  ib,  containing  the  serum  and  complement  but 
without  antigen  add  14  c.c.  salt  solution.  Incubate  for  absorption  of  complement. 
So  soon  as  these  tubes  are  placed  in  the  incubator  prepare  a  mixture  of  i%  washed 
red  cells,  according  to  the  amount  to  be  used  in  the  number  of  tests  to  be  carried 
out,  and  add  2  units  of  amboceptor  paper  for  each  Y%  c.c.  of  this  red-cell  emulsion. 
Incubate  along  with  the  other  tubes.  When  the  period  for  the  primary  incubation  is 
completed  add  to  each  tube  K  c.c.  of  the  emulsion  of  sensitized  red  cells.  ^ 

Incubate  again  and  readings  can  usually  be  made  in  ten  to  fifteen  minutes  and 
give  unusually  clear-cut  readings. 


184  PRACTICAL   METHODS  IN   IMMUNITY 

General  Considerations. — Cherry  thinks  anticomplementary  bodies  are  found 
during  chloroform  anaesthesia.  If  the  antigen  should  also  have  anticomplementary 
action  the  total  might  give  a  negative  result. 

By  heating  the  serum  for  half  an  hour  at  s6°C.  (inactivation)  the  positive  results 
reported  to  have  been  obtained  in  certain  cases  of  cancer,  nephritis,  scarlet  fever, 
leprosy  and  tuberculosis  may  be  avoided;  the  syphilitic  antibody  alone  being  more 
thermostable.  The  thermostability  of  serum  of  inherited  syphilis  is  the  highest — 
that  of  primary  syphilis  the  least  of  luetic  sera. 

McDonagh  states  that  in  the  primary  stage  the  Wassermann  is  positive  in  40%  of 
cases.  In  secondary  cases  97%  give  positive  results  when  treatment  has  not  been 
instituted.  In  tertiary  syphilis  about  70%  are  positive. 

In  268  cases  at  the  medical  clinic  of  Johns  Hopkins  Hospital,  Clough 
failed  to  obtain  a  positive  reaction  in  99  cases  which  were  negative 
clinically. 

In  45  cases  of  syphilis  he  obtained  73%  of  positive  results.  Ex- 
cluding cases  which  had  received  thorough  treatment  82%  were 
positive.  Tabes  gave  40%  and  general  paresis  100%.  In  five  cases 
of  primary  syphilis  four  gave  positive  reactions.  Kolmer  gives  96% 
positives  for  untreated  active  tertiary  syphilis  with  75  to  80%  for 
latent  tertiary  syphilis.  In  untreated  congenital  syphilis  of  children 
over  one  year  of  age,  97  to  100%.  Comparing  the  luetin  reaction 
with  the  Wassermann,  Noguchi  gives  80%  for  tertiary  and  70% 
positive  for  congenital  syphilis. 

Based  upon  the  observation  of  Bauer,  that  human  serum  contains  haemolytic 
amboceptors  for  sheep  corpuscles,  and  of  Hecht,  that  the  complement  normally 
present  in  human  serum  would  suffice  without  the  addition  of  guinea-pig  serum 
complement,  the  following  method  of  Fleming  is  easy  of  application,  but  not 
recommended. 

For  the  test  we  use: 

1.  Alcoholic  extract  of  rabbit's  heart,  made  by  washing  the  recently  removed 
heart  with  salt  solution  to  remove  all  blood.     Cut  into  small  pieces  and  grind  in  a 
mortar  with  sand  and  for  every  gram  of  heart  add  5  c.c.  of  95%  alcohol.     Keep  the 
mixture  at  a  temperature  of  6o°C.  for  two  hours  and  filter.     This  is  the  stock  solu- 
tion.    For  use  dilute  it  10  times  with  normal  salt  solution. 

2.  A  5%  emulsion  of  washed  sheep  red  cells,  prepared  as  for  the  Wassermann  test. 

3.  Suspected  and  control  sera. 

With  a  capillary  bulb  pipette  take  up  i  part  of  serum  and  4  parts  of  the  heart 
antigen,  mix  on  a  glass  slide,  again  draw  up  into  the  capillary  pipette  and, 
leaving  a  separating  air  space,  next  draw  up  i  part  of  5%  emulsion  of  sheep  red 
cells.  Then  seal  off  tip  of  pipette  and  incubate  at  37°C.  for  one  hour.  Now  file 
off  tip  and  mix  the  red  cells  with  the  serum  and  antigen  and  again  draw  up  into  the 
capillary  pipette  and  incubate  a  second  time  for  two  hours.  Haemolysis  or  the 
reverse  is  shown  in  the  fluid  overlying  the  cell  sediment.  Various  controls  should 
be  made  using  normal  and  known  syphilitic  sera;  also  with  normal  salt  instead  of 
serum. 


BACTERIAL  COMPLEMENT  FIXATION  TESTS  185 

The  objections  to  methods  using  human  serum  for  complement  are 
i.  the  great  variation  in  the  complement  content  of  different  human 
sera;  2.  human  complement  requires  about  10  times  as  much  ambo- 
ceptor  as  guinea-pig  complement  and  is  less  sensitive  to  fixation,  and 
3.  the  statement  is  made  by  some  workers  that  while  homologous 
complement  and  amboceptor  may  be  efficient  yet  the  complement  of  a 
serum  will  not  act  upon  its  homologous  antigen.  This  is  not  true 
because  the  complement  of  human  serum  invariably  haemolyzes  the 
homologous  antigen  (human  red  cells). 

The  various  precipitate  tests  that  have  been  proposed  are  unreliable.  The 
precipitate  reactions  with  bile  salts  give  better  results  than  with  lecithin,  this  latter 
showing  positive  results  in  almost  one-half  of  non-syphilitic  cases. 

BACTERIAL  COMPLEMENT-FIXATION  TESTS 

The  two  bacterial  complement-fixation  tests  which  are  used  as 
routine  diagnostic  methods  are  those  for  gonorrhoeal  and  glanders 
infections. 

Of  course  similar  tests  may  be  made  for  typhoid,  cholera,  etc.,  but  we  have  more 
simple  and  practical  methods  in  the  use  of  agglutination  or,  in  the  case  of  typhoid, 
blood  culture  procedures. 

The  two  best  known  methods  for  preparing  bacterial  antigens  are 
the  following: 

1.  Emulsify  the  growth  on  agar  or  starch  agar  (for  gonococcus)  in  salt  solution,  as 
described  under  preparation  of  vaccines.     Heat  the  emulsion  at  6o°C.  for  one  or  two 
hours  and  then  count  the  organisms  as  for  vaccines. 

For  gonococcus  tests  we  use  an  antigen  with  4,000,000,000  organisms  in  i  (?.c. 
This  may  be  used  directly  as  antigen  or  it  may  be  shaken  up  with  glass  beads  for 
several  hours  to  complete  disintegration.  The  antigen  can  be  preserved  by  the 
addition  of  y±%  trikresol  or  Y2%  phenol.  For  glanders  one  may  use  a  seventy- 
two-hour  culture  in  glycerine  bouillon,  sterilized  at  6o°C.  for  two  hours  and  preserved 
with  0.5%  phenol. 

2.  Besredka  and  Gay  prepare  their  antigen  by  precipitating  the  saline  bacterial 
emulsion,  washed-off  agar,  with  an  equal  amount  of  absolute  alcohol.     Then  centrifu- 
galize,  pipette  off  supernatant  fluid  and  dry  the  sediment  in  vacua  over  sulphuric 
acid.     The  dried  sediment  is  made  into  a  2%  suspension  with  isotonic  salt  solution. 
For  use  this  stock  solution  is  diluted.     There  are  also  methods  in  which  the  bacterial 
sediment  is  frozen  with  carbon  dioxide  snow  and  then  triturated  with  crystals  of 
sodium  chloride  so  as  to  make  an  isotonic  saline  emulsion. 

Bacterial  sediments  can  also  be  dried  in  calcium  chloride  desiccators. 

In  carrying  out  bacterial  complement-fixation  tests  we  use  an  amount 


1 86  PRACTICAL   METHODS   IN  IMMUNITY 


of  antigen  which  will  by  its  antigenic  power  alone  fix  complement  or, 
as  is  often  stated,  be  anticomplementary. 

Then  use  one-half  this  amount  as  the  antigen  content  for  the  test. 

The  method  of  Noguchi,  as  previously  described,  but  using  one-half  the  anticom- 
plementary dose  of  antigen,  is  satisfactory  after  experimenting  with  the  proper 
amount  of  inactivated  serum  of  the  patient  to  be  examined. 

For  the  Gonococcus  fixation  test  it  is  most  important  to  have  antigen 
prepared  from  a  mixture  of  several  strains  of  gonococci,  preferably 
10  or  12. 

In  our  laboratory  we  have  had  sharper  and  more  satisfactory  readings  by  employ- 
ing the  Emery  technic,  placing  in  the  first  antigen  tube  an  emulsion  containing 
4,000,000,000  organisms  to  the  c.c. 

It  is  also  satisfactory  to  have  the  antigen  in  tube  i  so  concentrated  that  it  will 
prove  anticomplementary.  Tube  2  would  contain  one-half  this  amount  of  antigen; 
tube  3,  one-fourth;  tube  4,  one-eighth;  tube  5,  one-sixteenth;  tube  6,  one-thirty-second; 
tube  7,  one-sixty-fourth.  Some  sera  are  strong  enough  in  specific  gonococcus  anti- 
body to  bring  about  complement  fixation  in  tube  8.  Along  with  the  test  of  the 
patient's  serum  we  should  carry  out  tests  with  known  negative  and  positive  sera. 

DETERMINATION  OF  OPSONIC  POWER  AND  THE  PREPARATION 
OF  VACCINES 

In  that  which  has  been  considered  in  the  previous  pages  only  the 
theories  of  Ehrlich  have  been  brought  out.  In  order  to  understand  the 
problems  involved  in  the  study  of  opsonins  the  phagocytic  theory  of 
immunity  brought  forward  by  Metchnikoff  must  be  studied.  Ehrlich's 
views  would  seem  to  hold  with  diseases  where  there  is  an  increase  in 
bacteriolytic  or  antitoxic  power  of  the  serum  while  in  such  diseases,  as 
are  caused  by  pathogenic  cocci,  the  pjiagocvtic  element  js  operative  as 
there  is  an  absence  of  bacteriolvtic  power  in  the  serum  of  the  person 
with  the  infection. 

There  are  two  kinds  of  phagocytes,  the  microphages  (represented  by  the  poly- 
morphonuclears)  which  on  phagolysis  or  disintegration  give  off  microcytase,  a 
bactericidal  substance.  Cytase  is  the  same  as  complement  or  alexine. 

The  microphages  are  chiefly  bactericidal  while  the  macrophages,  represented  by 
the  large  mononuclears  of  the  blood  and  fixed  connective-tissue  cells^exert  their 
action  on  protozoa  or  animal  cells.  . 

(t) 

Phagocytes  may  either  act  by  m^estin^  j^cteria  and  destroying 

them  intracellulary  or  they  may  as  a  result  of  phagolysis  bring  about 
bacteriolysis   exr.ra.r.p.11n1a.rlv.     According   to   Metchnikoff   the    intra- 


OPSONIC  INDEX  187 

cellular  bacteriolysis  explains  why  an  individual  may  possess  immunity 
and  yet  his  serum  fail  to  show  any  bacteriolytic  power. 

The  following  modification  of  Irishman's  method  takes  very  little  time  and  skill 
and  is  applicable  in  the  determination  of  the  organism  concerned  in  an  infection,  as 
in  Wright's  method.  The  control  of  vaccine  treatment  by  taking  opsonic  indices 
from  time  to  time  does  not  seem  to  have  met  with  much  favor  in  this  country — the 
sources  of  error  being  as  great  if  not  greater  than  ordinary  variations  in  the  opsonic 
index  during  the  negative  and  positive  phases. 

Method. — We  start  with  a  i  %  solution  of  sodium  citrate  in  salt  solution.  With 
this  emulsify  a  twelve-  to  twenty-four-hour  agar  slant  growth  of  the  organism  to  be 
tested  using  6  to  8  c.c.  of  the  citrated  salt  solution.  The  bacterial  emulsion  is  now 
poured  into  a  bottle  or  sealed  off  in  a  test-tube  and  shaken  thoroughly  in  a  shaker  or 
by  hand.  The  emulsion  is  then  centrifuged  to  throw  down  the  bacterial  clumps  and 
the  supernatant  slightly  turbid  bacterial  suspension  pipetted  off.  If  working  with  a 
dangerous  pathogen  it  is  advisable  to  kill  the  organisms  as  in  making  vaccines. 

Now  with  a  capillary  bulb  pipette  so  graduated  that  the  one  volume  mark  con- 
tains about  o.i  c.c.  we  draw  up  one  volume  of  citrated  salt  solution.  Then  having 
made  a  break  with  an  air  column,  we  take  up  one  volume  of  the  patient's  blood. 
Again  make  an  air  break  and  draw  up  one  volume  of  the  citrated  salt  solution 
bacterial  emulsion.  The  3  volumes  are  then  immediately  forced  out  into  a 
small  test-tube,  made  from  3  inches  of  ^6-inch  glass  tubing,  as  shown  in  the 
Emery  technic  for  the  Wassermann.  The  citrate  prevents  coagulation  of  the  blood 
and  the  contents  of  the  tube  are  well  mixed  by  drawing  up  and  ejecting  with  the 
capillary  bulb  pipette.  Incubate  this  small  test-tube  at  body  temperature  for  fifteen 
minutes,  shaking  the  contents  once  or  twice  during  the  incubation  period.  Exactly 
at  the  expiration  of  the  period  of  incubation  (usually  fifteen  minutes  although  at 
times  ten  minutes  or  thirty  minutes  may  be  desirable)  place  the  tube  in  a  centrifuge 
and  throw  down  the  cell  sediment.  Next  pipette  off  the  supernatant  fluid  and  then 
plunge  a  finely  drawn  out  capillary  pipette  to  the  bottom  of  the  tube  and  draw  off 
the  greater  part  of  the  sediment  at  the  bottom.  This  consists  largely  of  the  red 
cells  the  leukocyte  layer  on  the  surface  being  undisturbed. 

Now  mix  the  remaining  cell  sediment  and  smear  out  on  a  slide  or  preferably 
between  two  cover-glasses  as  in  Ehrlich's  method.  The  smear  is  fixed  by  burn- 
ing off  a  film  of  alcohol  or  with  methyl  alcohol  and  stained  with  dilute  carbol 
fuchsin  or  methylene  blue.  The  granule  staining  with  Wright's  stain  makes  it 
slightly  confusing. 

A  second  similar  preparation  but  using  blood  from  a  normal  person  as  a  control 
is  then  made.  Counting  the  phagocytized  bacteria  in  a  given  number  of  poly- 
morphonuclears,  we  obtain  an  average  number  of  bacteria  phagocytized  per  cell. 
Repeating  the  count  with  the  control  or  normal  blood,  we  likewise  have  the  average 
number  of  bacteria  taken  up  per  cell.  Dividing  the  patient's  average  by  the 
normal  average,  we  have  the  opsonic  index.  If  the  average  for  50  of  the  patient's 
cells  was  8  and  that  of  the  control  only  '4,  the  patient's  index  would  be  2,  or  twice 
the  normal.  The  practical  value  of  this  test  is  that  where  two  or  more  organisms 
are  on  a  plate  from  a  body  fluid  we  may  ascertain  the  causative  organism  by 
noting  marked  variation  from  the  normal  in  the  patient's  opsonic  index  for  that 


1 88  PRACTICAL   METHODS  IN   IMMUNITY 

particular  organism  and  not  for  the  other  organism.     This  variation  may  be  of 
the  nature  of  a  high  or  low  opsonic  index. 


METHOD  OF  WRIGHT  FOR  OBTAINING  OPSONIC  INDEX 

While  other  observers  had  previously  noted  the  presence  of  sub- 
stances in  immune  sera  which  so  acted  on  the  bacteria  that  phagocytosis 
was  made  possible,  yet  it  was  to  Wright  and  Douglas,  in  1903,  that  the 
existence  of  this  factor  in  phagocytosis  was  brought  forward  and  the 
estimation  of  such  substances  made  practicable. 

To  this  substance  the  name  opsonin  was  given — the  Greek  word  from  which  it  is 
derived  indicating  preparation  of  the  food — that  is,  the  opsonin  so  alters  or  sensitizes 
the  bacteria  that  they  can  be  engulfed  or  phagocytized  by  the  polymorphonuclear 
leukocytes  (the  microphages  of  Metchnikoff) .  About  the  same  time  Neufeld  and 
Rimpau  noted  the  presence  of  a  substance  in  immune  sera  which  so  acted  on  bacteria 
as  to  prepare  them  for  phagocytosis.  Their  designation  "bacteriotropic  substance" 
is  practically  synonymous  with  opsonin. 

In  1902  Leishman  introduced  the  method  of  determining  the  "phagocytic  index." 
By  taking  i  part  of  blood  and  impart  of  an  emulsion  of  the  bacteria  in  question  and 
keeping  the  mixture  in  a  moist  chamber  at  body  temperature  for  a  standard  time, 
as  fifteen  to  thirty  minutes,  and  then  spreading  the  blood-bacteria  mixture  and 
staining  the  film  with  Leishman  or  Wright's  stain  he  counted  the  number  of  bacteria 
in  a  certain  number  of  polymorphonuclears,  and  by  dividing  obtained  the  average 
number  per  leukocyte  of  bacteria  phagocytized. 

The  Wright  technic  for  determining  the  phagocytic  average,  and 
from  this  the  opsonic  index,  is  as  follows: 

Blood  is  taken  from  the  patient  and  at  the  same  time  from  a  normal  individual, 
or  preferably  the  blood  of  several  normal  individuals  is  pooled.  This  blood  is  best 
collected  in  a  Wright's  tube,  although  it  may  be  received  in  a  small  test-tube.  After 
coagulation  and  separation  of  the  serum,  the  serum  is  ready  for  use. 

The  next  step  is  to  prepare  the  leukocyte  gmulsion.  For  this  we  fiU  a  centri- 
fuge tube  with  normal  saltsolution,  to  which  has  been  added  i% 'sodium,  citrate 
— the  latter  to  prevenTcoagulation.  Then  having  pricked  a  finger  congested  by  a 
constricting  rubber  band,  from  15  to  20  drops  of  blood  are  added  to  the  citrated 
salt  solution,  and  the  mixture  thoroughly  shaken.  After  centrifugalization  for  about 
five  minutes  the  red  corpuscles  will  be  thrown  to  the  bottom  of  the  tube  with  the 
leukocytes  forming  a  superimposed  layer.  In  order  to  free  the  leukocytes  entirely 
from  serum  admixture,  the  supernatant  citrated  salt  solution  is  pipetted  off,  and  a 
fresh  tubeful  of  salt  solution  is  added  to  the  blood-cell  sediment.  Again  shak- 
ing, we  centrifuge,  obtaining  for  a  second  time  a  sediment  of  blood  cells  with  the 
leukocytes  in  the  superimposed  layer.  In  some  laboratories  the  washing  in  salt 
solution  is  again  repeated,  but  for  all  practical  purposes  two  washings  as  described 
above  suffice. 

The  superimposed  layer  of  vyhite  cells  may  now  be  pipetted  off  from  the  heavier 


VACCINES  189 

red  cells  (of  course,  containing  a  large  admixture  of  red  cells)  to  be  used  as  a  leuko- 
cyte cream — or  by  slanting  the  centrifuge  tube  we  can  pipette  off  the  proportion 
of  the  leukocyte  mixture  needed  from  the  bottom,  sides  or  top  of  the  slanted  layer 
of  blood  cells. 

Having  prepared  our  leukocyte  j;mulsion.  and  the  serum  from  the  normal  indi- 
vidual  as  well  as  that  from  the  patient,  it  only  remains  to  prepare  our  bacterial 
emulsion.  For  bacteria  in  general,  with  the  exception  of  tubercle  bacilli,  we  simply 
take  up  a  small  loopful  of  a  young  agar  culture  (eighteen  hours  or  less),  and  emulsify 
it  uniformly  with  salt  solution,  added  by  degrees  until  the  suspension  amounts  to 
Yz  to  i  c.c.,  and  giving  a  faint  turbidity.  To  thoroughly  distribute  and  especially 
to  break  up  clumps  repeated  suction  and  ejection  with  a  capillary  pipette  provided 
with  a  rubber  nipple  is  satisfactory. 

The  presence  of  clumps  in  a  bacterial  emulsion  invalidates  the  estimation  of 
phagocytosis,  for  the  reason  that  a  leukocyte  will  take  up  a  clump  of  twenty  or  more 
bacilli  as  readily  as  one  separate  organism. 

Having  at  hand  (i)  the  suspension  of  leukocytes,  (2)  the  bacterial  emulsion, 
and  (3)  the  sera  of  the  patient  and  the  normal  individual,  we  are  ready  to  proceed 
with  the  test. 

Using  a  capillary  bulb  pipette  with  a  pencil  mark  to  indicate  i  volume  we  draw 
up  to  the  mark  (i)  the  leukocyte  cream.  Then  wiping  off  the  tip  of  the  pipette  we 
draw  up  this  volume  of  leukocyte  emulsion  about  %  inch  to  make  an  air  break 
between  this  and  (2)  i  volume  of  the  bacillary  emulsion.  Again  making  an  air 
space  we  draw  up  (3)  the  serum  of  the  normal  individual.  This  gives  three  columns 
in  the  capillary  tube  with  intervening  breaks  of  air.  We  next  eject  the  three  con- 
stituents into  a  watch-glass  and  thoroughly  mix  them  by  alternate  suction  and  ejec- 
tion with  the  tube  and  nipple.  When  mixed  we  draw  the  mixture  up  into  the  same 
capillary  tube,  seal  off  the  capillary  end  in  the  flame  and  put  in  an  incubator  for 
exactly  fifteen  minutes. 

We  next  repeat  the  process  identically  except  that  the  patient's  serum  is  used  in- 
stead of  that  of  the  normal  individual. 

These  tubes  having  been  kept  at  the  same  temperature  for  the  same  length  of 
time  are  then  taken  out,  the  contents  blown  into  a  watch-glass,  mixed  thoroughly 
a  second  time,  and  then  a  smear  is  made — a  drop  of  the  mixture  being  deposited 
on  a  very  clean  slide  and  the  smear  made  by  a  second  narrower  slide  (by  cutting  off 
the  corner  of  the  slide)  which  is  drawn  along  in  a  zigzag  way.  The  smears  are  then 
stained  (Leishman's  or  Wright's  blood  stain  or  Ziehl-Neelson's  for  tubercle  bacilli) 
and  the  number  of  the  bacteria  in  from  50  to  100  leukocytes  counted.  This 
number  divided  by  the  number  of  cells  gives  the  phagocytic  average. 

The  phagocytic  average  of  the  patient's  tube  divided  by  that  of  the  normal  in- 
dividual's tube  gives  the  opsonic  index.  Thus,  in  counting  100  cells  we  find  500 
phagocytized  cocci  in  the  patient's  tube,  giving  an  average  of  5,  and  in  the  normal 
individual's  blood  we  get  1000,  an  average  of  10.  Then  the  opsonic  index  would  be 
5  -f-  10,  or  0.5. 

PREPARATION  or  VACCINES 

It  has  been  found  satisfactory  to  make  use  of  stock  vaccines  in- 
gonorrhceal  and  tuberculous  affections.  In  treatment  of  tuberculosis 


1  go        '  PRACTICAL   METHODS  IN  IMMUNITY 

Wright  prefers  Koch's  T.  R.  or  Neu  Tuberculin  in  doses  of  from  J 
to  J^oo  rng.  Some  prefer  Koch's  more  recent  bazillen  emulsion.  In 
case  of  other  infections,  however,  and  preferably  with  gonorrhceal  in- 
fections, the  causative  organism  should  be  isolated  from  pus,  sputum, 
urine,  blood,  or  other  material  from  patient  (autogenous  vaccine). 

In  the  making  of  vaccines  all  media  and  apparatus  should  be  sterilized  with 
scrupulous  care  to  avoid  the  danger  of  tetanus  infection.  Having  isolated  the  organ- 
ism, it  is  inoculated  upon  one  or  more  agar  slants,  and  after  a  growth  of  from  five 
to  seven  hours  with  streptococci  and  pneumococci,  or  with  eighteen  hours  for  staphy- 
lococci  and  colon,  the  growth  on  these  inoculated  slants  is  taken  up  with  salt  solu- 
tion, thoroughly  shaken  up  in  the  diluting  solution  and  standardized.  (Esmarch 
roll  of  nutrient  agar  may  be  inoculated  for  growing  cultures  for  vaccines.) 

The  most  practical  way  is  to  gently  rub  off  the  growth  on  the  agar  in  about  i  or 

2  c.c.  of  salt  solution  with  a  platinum  loop  or  sterile  cotton  swab.    Then  pour  the 
bacterial  emulsion  into  a  sterile  test-tube  and  repeat  the  process  with  three  to  five 
agar  slants,  until  we  have  from  6  to  10  c.c.  of  the  emulsion  in  the  sterile  test-tube. 
By  heating  to  melting-point  in  the  flame  a  piece  of  glass  tubing  and  attaching  it  to 
the  rim  of  the  test-tube  (also  melted),  we  have  a  handle  with  which  to  draw  out  the 
test-tube  when  heated  about  i  inch  from  the  mouth  in  a  blowpipe  flame.     Drawing 
this  out,  we  let  it  cool,  and  then  filing  the  constricted  portion  we  break  it  off  and  seal 
it  in  the  flame.     By  shaking  up  and  down  vigorously  for  five  to  fifteen  minutes,  or 
preferably  in  a  mechanical  shaker  the  bacteria  are  distributed  evenly  in  the  salt 
solution.     A  piece  of  platinum  wire,  twisted  into  corkscrew  shape,  and  fused  in  the 
drawn-out  end  of  the  containing  test-tube  helps  in  breaking  up  the  bacterial  emulsion 
and  is  a  great  aid  in  the  preparation  of  streptococci  or  diphtheroid  vaccines. 

The  sealed  test-tube  is  then  placed  in  a  water-bath  at  6o°C.  and  heated  at  this 
temperature  for  one  hour.  Again  shake.  The  constricted  sealed  end  is  again  filed 
off  and  a  few  drops  shaken  out  in  a  watch-glass  for  standardization,  and  at  the  same 
time  a  few  drops  are  deposited  on  an  agar  slant  as  a  test  for  sterility.  (Incubation 
for  twenty-four  to  forty-eight  hours  should  not  show  growth.) 

Wright  found  that  by  taking  a  definite  quantity  of  blood  and  a 
similar  quantity  of  bacterial  emulsion,  mixing  the  blood  and  bacterial 
emulsion,  then  making  a  smear  and  staining,  it  was  possible  to  de- 
termine the  ratio  of  bacteria  to  red  cells,  and  from  this  the  number  of 
bacteria  per  cubic  centimeter  could  be  determined.  For  example,  if 
we  find  three  bacteria  to  each  red  cell  we  should  have  15,000,000 
bacteria  to  i  c.mm.  (There  being  5,000,000  red  cells  to  the  cubic  milli- 
meter.) As  i  c.c.  is  1000  times  greater  than  i  c.  mm.,  there  would 
be  15,000,000,000  bacteria  in  each  c.c.  of  such  an  emulsion,  or  vaccine, 
as  it  is  termed. 

The  standardization  made  with  a  haemacytometer  is  best  done  by  drawing  up 
the  vaccine  to  0.5  with  either  the  red  or  white  pipette,  according  to  concentration, 


DOSAGE  OF  VACCINES  igi 

and  then  sucking  up  one- twentieth  of  i%  dahlia  in  i%  formalin  to  n  or  101. 
Allow  the  bacteria  to  settle  on  the  shelf  for  ten  minutes  before  counting.  Count  as 
in  making  a  red  count. 

The  use  of  a  piece  of  amber  glass  in  front  of  an  incandescent  light 
enables  one  to  pick  up  the  bacteria  more  satisfactorily  as  well  as  to 
differentiate  bacteria  from  debris.  A  counting  chamber  with  a  depth 
of  J^o  mm.  is  to  be  preferred  to  the  ordinary  %  o  mm.  chamber  as  one 
can  begin  the  count  after  about  five  minutes  time  for  settling. 

A  satisfactory  diluting  fluid  is  that  recommended  by  Callison.  It  is :  Hydrochloric 
acid  2  c.c.,  Bichloride  of  mercury  (i  to  500  aq.  sol.)  100  c.c.,  and  sufficient  i% 
aqueous  solution  of  acid  fuchsin  to  color  the  diluting  mixture  a  deep  cherry  red. 
The  diluting  fluid  should  then  be  filtered.  The  bichloride  forms  an  albuminate 
on  the  surface  of  the  bacteria  which  promotes  rapid  sedimentation  and  the  fuchsin 
stains  the  bacteria. 

Having  determined  the  strength  of  the  stock  vaccine,  we  should 
prepare  a  dilute  vaccine  for  injection. 

This  is  most  conveniently  carried  out  by  filling  vials  with  50  c.c.  of  salt  solution, 
plugging  with  cotton,  then  sterilizing  in  the  autoclave.  A  sterile  rubber  cap  is  now 
drawn  over  the  mouth  of  the  vial.  Sterility  is  insured  by  plunging  the  rubber  cap 
and  neck  in  boiling  water.  If  the  stock  vaccine  showed  5,000,000,000  bacteria  per 
c.c.  and  we  desired  to  have  a  vaccine  containing  200,000,000  bacteria  per  c.c.,  it 
would  be  necessary  to  draw  out  2  c.c.  of  the  salt  solution  by  means  of  a  sterile  syringe 
needle  inserted  through  the  rubber  cap  and  replace  it  with  2  c.c.  of  the  bacterial 
emulsion.  Example:  In  introducing  2  c.c.  of  a  vaccine  containing  5,000,000,000 
bacteria  per  c.c.,  we  throw  in  10,000,000,000  bacteria  in  a  volume  equal  to  50  c.c. 
Then  each  c.c.  of  the  50  c.c.  in  the  bottle  would  contain  10,000,000,000  divided  by 
50  or  200,000,000  in  each  c.c.  If  we  only  want  a  vaccine  containing  100,000,000  per 
c.c.  we  should  only  add  i  c.c.  We  now  add  Y±%  of  trikresol  to  the  vaccine  in  order 
to  insure  sterility.  (Introduced  with  syringe,  inserting  needle  through  rubber  cap.) 
The  syringe  is  best  sterilized  by  drawing  up  vaseline  or  olive  oil  heated  to  i5o°C., 
and  the  neck  and  rubber  cap  of  the  bottle  in  boiling  water.  We  now  draw  up  the 
desired  dose  of  bacteria.  If  glass  syringes  are  used,  simply  boiling  in  water  suffices. 
One  can  purchase  ampoules  which  are  sterilized  and  filled  with  a  standardized 
emulsion.  They  are  sealed  in  the  flame  and  labelled  with  the  bacterial  content 
per  cc. 

The  ordinary  doses  are:  For  gonococci,  streptococci,  pneumococci, 
and  colon  vaccines,  5,000,000  to  50,000,000.  For  staphylococci 

200,000,000  tO  1,000,000,000. 

Wilson  gives  the  following  minimum  and  maximum  doses  expressed  in  millions: 
Streptococcus,  6  and  68. 
Gonococcus,  45  and  900. 
Meningococcus,  300  and  900. 


I Q2  PRACTICAL   METHODS  IN  IMMUNITY 

M.  mclilensis,  700  and  1400. 

B.  coli,  1 6  and  240. 

B.  typhosus  (treatment)  100  and  250. 

B.  typhosus  (prophylaxis)  500  and  1000. 

B.  pyocyanens,  34  and  1000. 

B.  pneumonia,  44. 

Staphylococci,  150  and  900. 

B.  tuberculosis,  Hoooo  to  /^oo  rag. 

Sensitized  Vaccines. — These  are  prepared  by  treating  the  bacteria 
with  the  specific  serum  and  cause  less  reaction  than  ordinary  vaccines. 
To  prepare  them  the  bacterial  growth  is  treated  with  its  antiserum 
for  three  hours,  thrown  down  in  the  centrifuge  and  the  supernatant 
serum  removed.  After  washing  in  salt  solution  they  are  emulsified  in 
salt  solution  and  killed  at  a  temperature  of  56°C.  for  one  hour.  Besredka 
has  used  living  sensitized  bacteria  in  typhoid. 

The  question  of  the  best  method  of  preparing  vaccines  for  prophylactic  use  is  still 
unsettled.  The  greatest  difficulty  has  been  experienced  in  making  vaccines  of  the 
Shiga  bacillus  on  account  of  the  great  toxicity  of  such  preparations. 

Thompson  has  recently  carried  out  some  very  important  experiments  at  the 
Lister  Institute. 

He  worked  with  vaccines  heated  to  s6°C.  for  one  hour  using  ordinary  methods  as 
well  as  organisms  sensitized  by  treatment  with  specific  serum.  In  another  series 
of  vaccines  he  sterilized  ordinary  cultures  as  well  as  sensitized  ones  with  0.5% 
carbolic  acid  in  normal  saline.  He  found  that  sterilfzation  by  heat  not  only  destroyed 
much  of  the  immunizing  power  of  the  vaccines,  but  that  such  vaccines,  whether  of 
ordinary  bacterial  emulsions  or  of  sensitized  organisms,  showed  great  toxicity  upon 
their  being  injected  and  the  heated  sensitized  ones  were  somewhat  more  toxic  than 
the  nonsensitized  organisms.  Dean  has  used  "eusol"  for  Shiga  vaccines. 

On  the  whole  it  would  seem  that  sterilization  with  %%  carbolic  or  Y±%  trikresol, 
using  ordinary  bacterial  emulsions,  is  better  than  other  methods. 

Of  course  living  organisms  subjected  to  their  specific  serum  have  been  recom- 
mended in  the  case  of  typhoid  but  such  methods  are  certainly  not  devoid  of  danger 
and  are  not  to  be  recommended  for  the  present. 

ANAPHYLAXIS 

This  is  a  term  which  indicates  the  opposite  of  prophylaxis.  It  was 
noted  that  after  a  period  of  incubation  of  at  least  ten  days  a  second 
injection  of  horse  serum  produced  symptoms  of  respiratory  embarrass- 
ment, convulsions  and,  at  times,  death.  The  primary  injection  had 
during  the  period  of  incubation  sensitized  the  cells  to  this  particular 
proteid.  Extremely  small  amounts  of  serum  will  sensitize  (o.oooi  c.c.). 
For  anaphylaxis  production  much  larger  amounts  are  required;  from 


ANAPHYLAXIS 


193 


o.oi  to  0.3  c.c.  when  given  intravenously  or  intracardially  and  i  or  2  c.c. 
when  given  subcutaneously. 

This  phenomenon  of  sensitization  in  the  case  of  rabbits  bears  the 
name  of  Arthus,  and  as  applied  to  guinea-pigs  sensitized  with  diphtheria 
antitoxin  sera  the  name  Theobald  Smith,  and  it  is  stated  by  Muir 
and  Ritchie  that  active  research  as  to  anaphylaxis  may  be  said  to  date 
from  the  discovery  of  the  phenomenon  of  Theobald  Smith. 

Rosenau  and  Anderson  working  with  guinea-pigs  showed  that  small 
doses  were  efficient  for  sensitization,  that  the  condition  was  trans- 
missible from  mother  to  offspring  and  that  a  second  animal  could  be 
sensitized  by  being  injected  with  the  serum  of  a  sensitized  animal. 

This  group  of  symptoms,  the  so-called  anaphylactic  shock,  which  is  apt  to  set  in 
within  a  few  minutes  after  the  second  injection,  is  often  preceded  by  restlessness 
and  great  excitement  and  together  with  the  dyspnceic  manifestations  such  as  cough- 
ing and  rapid  breathing,  there  is  cardiac  weakness  and  great  fall  of  blood  pressure. 
The  more  serious  symptoms  as  convulsions  and  at  times  death  in  from  a  few  minutes 
to  an  hour  are  more  apt  to  appear  after  intracerebral  injections  than  after  intra- 
peritoneal.  Subcutaneous  injections  are  least  apt  to  produce  anaphylactic  symp- 
toms. Our  attention  to  this  phenomenon  commenced  with  the  study  of  "serum 
sickness"  or  "serum  disease."  In  this  an  erythematous  rash  or  urticaria  associated 
with  more  or  less  oedema  comes  on  after  eight  to  twelve  days  from  the  time  of  the 
first  and  only  injection  of  horse  serum.  It  is  supposed  to  be  due  to  the  fact  that 
some  of  the  serum  originally  injected  remains  unchanged  in  the  tissues  so  that 
when  the  sensitization  takes  place  there  is  present  and  at  hand  the  same  foreign 
proteid  to  bring  about  anaphylactic  symptoms. 

Immunization  against  anaphylaxis  is  possible  by  repeating  injection  of  the 
sensitizing  serum  or  proteid  during  the  period  of  incubation.  When  a  sensitized 
animal  recovers  from  the  second  injection  it  is  afterward  immune  to  anaphylaxis. 
This  is  termed  antianaphylaxis. 

It  is  important  to  note  that  this  hypersusceptibility  appears  to  be 
very  rarely  of  importance  in  the  matter  of  the  administration  of  a 
second  injection  of  diphtheria  antitoxin  after  the  period  of  anaphylactic 
incubation. 

As  a  rule  the  death  or  untoward  effects  of  the  injection  of  serum  are 
in  cases  of  status  lymphaticus.  Cases  in  man  do  occur,  however,  but 
with  extreme  infrequency,  in  which  within  a  few  minutes  after  the 
only  injection  of  serum  the  patient  becomes  restless,  shows  symptoms 
of  cardiac  and  respiratory  embarrassment  and  may  be  dead  in  a  very 
short  time. 

According  to  Rosenau  and  Anderson  individuals  who  have  asthmatic  tendencies 
as  well  as  those  who  have  had  serum  injections  ten  to  twelve  days  or  longer  prior 
13 


I94  PRACTICAL   METHODS  IN   IMMUNITY 

to  the  second  injection  should  be  considered  as  possible  subjects  for  anaphylactic 
shock. 

Vaughan  recommends  that  when  this  is  to  be  feared  one  should  only  give  about 
o.i  c.c.  of  the  serum  and  after  an  interval  of  two  hours,  provided  no  untoward 
symptoms  set  in,  to  give  the  full  amount  of  the  injection.  Besredka  advises  heating 
the  serum  to  56°C.  as  a  guard  against  anaphylactic  shock. 

Allergy. — The  condition  of  hypersusceptibility  or  anaphylaxis  is  at 
times  termed  allergy.  Thus  in  a  person  who  has  been  successfully 
vaccinated  a  reaction  shows  at  the  site  of  inoculation  within  twenty- 
four  hours  which  does  not  appear  in  the  nonimmune  person  for  a  period 
two  or  three  times  as  long.  The  diagnostic  tests  with  tuberculin  and 
luetin  are  hence  often  referred  to  as  allergic  reactions. 

Toxic  Protein  Split  Products. — It  may  here  be  stated  that  some  in- 
vestigators are  of  the  opinion  that  our  views  not  only  as  to  immunity 
but  as  to  the  essential  nature  of  infectious  diseases  may  be  later  on 
found  to  rest  in  production  of  anaphylaxis.  According  to  Vaughan 
and  others  the  parenteral  introduction  (hypodermic  as  opposed  to 
alimentary  tract  or  enteral  introduction)  of  foreign  proteids  excites  the 
formation  of  specific  ferments  in  the  cells  and  fluids  of  the  animal 
injected.  After  the  ferment  is  formed  a  second  injection  of  the  same 
proteid  activates  the  ferment  which  splits  up  the  proteid  into  a  poison- 
ous and  nonpoisonous  portion,  the  former  causing  the  symptoms  of 
anaphylaxis  or  disease  in  the  case  of  the  poisonous  split  proteid  of 
bacterial  pathogens. 

The  name  anaphylactine  has  been  applied  to  the  sensitizing  substance 
produced  during  the  period  of  incubation,  and  anaphylatoxin  to  the 
poisonous  part  of  the  split  proteid. 

•  It  has  been  proposed  to  employ  this  phenomenon  as  a  diagnostic  measure.  By 
taking  the  serum  of  a  tuberculous  patient,  which  would  contain  the  sensitizing  sub- 
stance, and  injecting  it  into  the  peritoneal  cavity  of  a  rabbit,  the  animal  would  be 
sensitized  and  an  injection  of  tuberculin  a  few  hours  later  would  bring  about  the 
phenomena  of  anaphylaxis  in  the  rabbit. 

This  passive  anaphylaxis,  as  it  is  termed,  usually  requires  approximately  twenty- 
four  hours  for  sensitization.  This  passive  anaphylactic  sensitization  seems  to  dis- 
appear in  two  weeks.  It  has  been  advised  to  passively  sensitize  guinea-pigs  with 
the  serum  of  the  person  about  to  be  injected  and  then  twenty-four  hours  after  inject 
the  guinea-pigs  with  the  curative  serum.  If  untoward  results  occur  in  the  guinea- 
pigs  the  patient  should  not  receive  the  injection. 

Recently  Hagemann  has  found  the  following  technic  valuable  in  the  diagnosis 
of  surgical  tuberculosis.  Guinea-pigs  are  inoculated  intraperitoneally  with  tuber- 
culosis cultures  and  by  the  end  of  the  second  week  such  pigs  are  sensitized.  The 
suspected  material,  as  serous  effusion,  is  injected  intracutaneously  and  within  twenty- 
four  to  forty-eight  hours  a  distinct  swelling  of  the  skin  with  a  bluish-red  center, 


ABDERHALDEN  TEST  195 

which  is  surrounded  by  a  porcelain  white  ring  and  outside  of  this  a  zone  of  inflam- 
mation, shows  a  positive  test. 

THE  ABDERHALDEN  REACTION 

According  to  Abderhalden,  specific  ferments  of  a  protective  nature 
(abwehrfermente)  appear  in  the  circulation  following  parenteral  injec- 
tion of  various  materials  and  are  different  from  ordinary  antibodies. 

The  substance  which  causes  the  production  of  these  specific  ferments 
is  called  the  substratum  instead  of  antigen,  the  usual  designation  of 
antibody  stimulating  substances.  The  principle  of  the  test  is  that 
when  serum  containing  such  specific  ferments  is  placed  in  contact,  in 
•vitro,  with  the  substratum  the  latter  is  digested  with  the  production  of 
soluble  products,  which  can  pass  through  a  dialyzer  and  be  recognized 
in  the  dialysate.  The  special  dialyzers  are  called  thimbles  and  those 
prepared  by  Schleicher  and  Schull  are  recommended. 

About  0.5  gram  of  the  substratum  is  placed  in  a  thimble,  which  has  been  intro- 
duced into  a  clean  Erlenmeyer  flask  and  covered  with  1.5  c.c.  of  the  patient's  serum. 

The  thimble  is  then  withdrawn,  its  open  end  closed  by  forceps,  then  thoroughly 
washed  with  distilled  water  and  again  introduced  into  a  clean  Erlenmeyer  flask 
containing  20  c.c.  of  sterile  distilled  water.  Toluol  is  then  introduced  into  the 
flask  so  as  to  cover  the  water  around  the  thimble.  These  flasks  are  then  put  in 
the  incubator  for  eighteen  hours  and  the  dialysate  tested  for  protein  by  the  ninhydrin 
test.  For  this  one  uses  0.2  c.c.  of  a  i%  solution  of  ninhydrin  in  water  and  adds 
10  c.c.  of  dialysate  and  the  mixture  brought  to  a  boil  in  a  test-tube.  A  positive 
reaction  is  a  violet  blue. 

The  biuret  reaction  may  also  be  used. 

The  difficulties  in  the  way  of  handling  the  thimbles  and  preparing  the  substratum 
are  so  great  that  Abderhalden  questions  the  competency  of  even  experienced 
serologists  for  the  carrying  out  of  the  test. 

The  main  objection  to  the  test,  however,  is  that  it  is  affected  quanti- 
tatively so  that  using  varying  amounts  of  reagents  one  may  obtain 
contradictory  results;  thus  it  is  possible  to  obtain  ninhydrin  reactions 
with  the  serum  of  a  male  animal  acting  upon  a  placental  substratum. 

While  the  test  is  best  known  in  connection  with  the  recognition  of 
pregnancy  it  has  also  been  employed  for  the  diagnosis  of  malignant 
tumors,  etc. 

In  taking  serum  for  the  test  it  is  necessary  to  withdraw  the  blood  as 
long  after  a  meal  as  possible,  preferably  in  the  morning  before  breakfast. 
The  blood  is  taken  into  paraffin-coated  centrifuge  tubes  and  most 
thoroughly  centrifuged,  so  that  it  is  absolutely  free  from  red  cells. 
It  is  pipetted  off  and  kept  on  ice. 


196  PRACTICAL   METHODS  IN  IMMUNITY 

For  the  substratum  the  placenta  should  be  obtained  as  soon  after 
delivery  as  possible  or  the  malignant  tumors  as  soon  after  operation 
as  is  feasible.  All  connective  tissue,  blood,  etc.,  should  be  removed 
from  the  tissue  from  which  the  substratum  is  to  be  prepared. 

The  tissue  is  then  cut  up  finely,  put  in  a  deep  jar  and  washed  several  times  with 
distilled  water.  It  is  then  boiled  in  a  great  excess  of  distilled  water — 100  times  as 
much — and  boiling  continued  about  thirty  minutes.  This  water  is  decanted  and 
the  tissue  added  to  fresh  boiling  water,  repeating  the  process  five  or  six  times  until 
when  placed  in  a  dialyzing  thimble  no  ninhydrin  reacting  soluble  proteins  can  be 
recognized  in  the  dialysate  by  the  blue  color. 

The  sterile  tissue  is  now  placed  in  a  jar,  covered  with  distilled  water  and  on  top 
of  this  toluol  is  deposited  to  maintain  the  sterility.  To  estimate  the  proper  amount 
of  substratum  to  use  in  a  test  one  must  determine  this  using  normal  as  well  as 
specific  sera,  recognizing  the  quantitative  error  of  the  test.  The  amount  usually 
called  for  is  0.5  gram. 

Bronfenbrenner  is  of  the  opinion  and  has  supported  it  by  much 
experimental  work  that  it  is  not  digestion  of  the  substratum  which 
takes  place  but  autodigestion  of  the  specific  serum  when  placed  in 
contact  with  its  substratum,  the  products  of  such  autodigestion  being 
of  the  nature  of  anaphylatoxins. 

Anaphylatoxin  Production  Test.  Bronf  enbrenner's  Modification  of 
Abderhalden's  Test. — Bronfenbrenner  found  that  if  he  treated  the 
serum  of  an  •animal  with  a  particular  substratum  and  then  injected 
the  autodigested  serum  intradermically  he  obtained  a  skin  reaction. 
This  reaction,  however,  only  obtained  for  homologous  animals,  those 
of  a  different  species  not  reacting  satisfactorily. 

He  now  reports  satisfactory  results  with  the  following  technic:  About  2  c.c.  of 
the  patient's  serum  is  injected  intraperitoneally  into  a  guinea-pig,  thus  passively 
transferring  to  the  guinea-pig  the  specific  substances  of  the  human  patient's  serum. 
The  next  day  the  guinea-pig  is  bled,  its  serum  collected  and  placed  on  ice  with  a 
suitable  amount  of  substratum  (placenta,  bazillen  emulsion,  tumor  tissue,  etc.). 

Eighteen  hours  later  the  serum  is  separated  from  the  substratum  by  centrifugaliza- 
tion,  pipetted  off  and  placed  in  the  incubator  for  fifteen  hours.  At  this  time  inject 
0.05  c.c.  of  the  autodigested  serum,  possibly  containing  anaphylatoxin,  into  the 
shaven  skin  of  a  normal  guinea-pig. 

From  twelve  to  twenty-four  hours  later  a  distinct  skin  reaction  occurs  at  the  site 
of  injection,  provided  the  serum  of  the  patient  contained  antibodies. 

Instead  of  the  skin  reaction  one  may  inject  0.5  c.c.  of  such  autodigested  serum 
into  the  heart  or  vein  of  a  normal  guinea-pig  and  death  will  result  of  anaphylactic 
shock. 

If  the  patient's  serum  were  negative  the  injection  of  as  much  as  5  c.c.  would  not 
cause  symptoms. 


PART  II 
STUDY  OF  THE  BLOOD 

CHAPTER  XIII 
MICROMETRY  AND  BLOOD  PREPARATIONS 

MlCROMETRY 

IN  the  examination  of  blood  and  faeces  preparations,  especially 
when  the  identification  of  animal  parasites  is  in  question,  there  is  noth- 
ing that  assists  more  than  a  knowledge  of  the  measurements  of  the 
object  studied.  The  making  of  such  measurements  microscopically  is 
termed  micrometry. 

Micrometry  is  also  indispensable  in  bacteriology  and  cytodiagnosis 
as  well  as  in  animal  parasitology. 

The  most  practical  way  of  making  these  measurements  is  with  an  ocular  microme- 
ter. These  can  be  bought  separately,  or  a  glass  disc  (disc  micrometer)  with  lines 
ruled  on  it  can  be  dropped  into  the  ocular  to  rest  on  the  diaphragm  inside  the  ocular. 
The  ruled  surface  of  this  glass  diaphragm  should  be  placed  downward.  As  was 
stated  in  connection  with  the  microscope,  the  image  of  the  object  is  formed  at  the 
level  of  the  diaphragm  rim  inside  the  ocular,  consequently  the  lines  of  the  image  cut 
those  of  the  lines  ruled  on  the  glass  in  the  ocular.  Once  having  standardized  the 
value  of  the  spaces  of  the  ocular  micrometer  for  each  different  objective,  all  that  is 
necessary  subsequently  in  measuring  is  to  count  the  number  of  lines  or  spaces  which 
the  image  of  the  object  fills  and  then,  knowing  the  value  of  each  space  for  that  ob- 
jective, to  multiply  the  number  of  spaces  by  the  value  of  a  single  space. 

The  Micron. — The  unit  in  micrometry  is  the  micron.  This  is  usually 
written  ju  and  is  the  Ho 00  Part  of  a  millimeter.  There  are  1000  microns 
in  a  millimeter. 

To  standardize:  For  this  purpose  it  is  necessary  to  have  a  scale  of  known  measure- 
ments. The  stage  micrometers  are  usually  ruled  in  spaces  of  o.i  and  o.oi  mm.  The 
lines  which  are  Ho  mm.  apart  are  consequently  separated  by  a  distance  of  100 
microns;  those  Koo  mm.  apart  are  separated  by  a  distance  of  10  microns. 

Ocular  Micrometer. — The  ocular  micrometer  is  usually  ruled  with 
50  or  100  lines  or  spaces,  separated  by  longer  lines  into  groups  of  5 
and  10. 

197 


MICROMETRY   AND  BLOOD   PREPARATIONS 


Having  brought  the  lines  on  the  stage  micrometer  to  a  focus,  we  determine  the 
number  of  spaces  on  the  stage  micrometer  which  the  50  divisions  of  the  ocular 
micrometer  cover.  To  distinguish  the  ruling  of  the  ocular  from  that  of  the  stage 
micrometer,  revolve  the  ocular  with  the  fingers. 

The  tube  length  which  is  used  at  the  time  of  standardizing  must 
always  be  adhered  to  in  subsequent  measurements. 


FIG.  51. — Micrometry  diagrams,  i.  Ocular  micrometer  with  stage  micrometer. 
50  spaces  of  ocular  micrometer  cover  two  zoo-micron  spaces  and  ten  lo-micron  spaces; 
equal  300  microns.  Each  division  on  ocular  micrometer  equals  6-microns.  2. 
Ocular  micrometer  subtending  image  of  whip-worm  egg.  9  spaces  of  ocular  mi- 
crometer cover  whip-worm  egg.  Each  space  equals  6  microns.  Whip-worm  egg 
equals  54  microns.  3.  Ocular  micrometer  with  ruling  of  hsemacytometer.  50 
spaces  of  ocular  micrometer  cover  space  equal  to  width  of  6  small  squares  50X6  =  300 
microns.  Each  division  of  ocular  micrometer  equals  6  microns. 

Example:  With  a  %-inch  objective,  the  50  rulings  of  the  ocular  micrometer 
fill  in  fifteen  of  the  ^0-mm.  rulings  (loojx)  and  three  of  the  Hoo-mm.  spaces  (io/z). 
Consequently  the  50  spaces  of  the  ocular  cover  1530  microns  (15  X  100  =  1500; 
3  X  10  =  30).  Then  if  50  spaces  equal  1530  microns,  one  space  would  equal 
30.6  microns.  With  the  J^-inch  objective  the  50  ocular  spaces  would  cover  about 
three  of  the  J^f  o  mm.  (ioo/u)  spaces  of  the  stage  micrometer.  Then  the  50  spaces 
would  equal  300  microns  and  one  space  would  equal  6  microns. 

The  ruling  of  the  slide  of  a  Thomas-Zeiss  haemocytometer  will  answer  as  well 
as  a  stage  micrometer.  The  small  squares  are  %Q  mm.  square,  consequently 


BLOOD   PREPARATIONS  I 99 

the  distance  between  the  lines  bordering  the  small  square  is  J^o  mm.  or  5° 
microns. 

Now,  if  with  the  ^-inch  objective,  the  50  lines  on  the  ocular  fill  in  the  spaces  of 
six  small  squares,  the  length  of  such  a  space  would  be  50  X  6  =  300  microns.  This 
divided  by  50  spaces  would  equal  6/*. 

Should  there  be  100  spaces  on  the  ocular  micrometer  instead  of  50,  it  would 
only  be  necessary  to  divide  the  length  in  microns  of  the  ruled  surface  of  the  stage 
micrometer  covered  by  the  100  lines  of  the  ocular  micrometer  by  100.  The  quotient 
would  give  the  value  in  microns  of  each  space  of  such  an  ocular  micrometer. 

Filar  Micrometer. — The  most  accurate  instrument  for  measuring  is  the  filar  mi- 
crometer. These  are  expensive.  Measurements  can  also  be  made  with  the  camera 
lucida,  but  it  takes  considerable  time  to  make  the  adjustments  necessary,  so  that  it 
is  not  convenient.  With  an  ocular  micrometer  one  can  make  measurements  of 
blood-cells,  amoebae,  etc.,  in  a  few  seconds — it  only  being  necessary  to  slip  in  the 
ocular  micrometer. 

Rule  for  determining  the  magnifying  power  of  microscopic  lenses:  Measure  the 
diameter  of  the  lens  of  the  objective  in  inches — the  approximate  equivalent  focal 
distance  is  about  twice  the  diameter.  Dividing  10  by  the  equivalent  focal  distance 
gives  the  magnifying  power  of  the  lens.  This  should  be  multiplied  by  the  number 
of  times  the  ocular  magnifies.  Example:  The  diameter  of  the  lens  of  the  objective 
was  found  to  measure  Y^  inch,  the  focal  distance  would  then  be  about  i  inch.  Divid- 
ing 10  by  i  we  have  10  as  the  magnifying  power  of  the  lens  of  the  objective.  If  we 
were  using  a  No.  4  ocular,  the  magnifying  power  would  be  approximately  forty. 

BLOOD  PREPARATIONS 

To  obtain  blood,  except  for  blood  cultures,  use  either  a  platino- 
iridium  hypodermic  needle  which  can  be  sterilized  in  the  flame,  a  small 
lancet,  or  a  surgical  needle  with  cutting  edge. 

When  using  such  surgical  needles  it  is  a  good  plan  to  sharpen  the  cutting  edge  on 
a  fine-grained  whetstone.  Afterward  the  needle  should  be  sterilized  by  boiling. 
Sterilization  of  a  needle  in  the  flame  blunts  the  cutting  edge.  A  steel  pen  with  one 
nib  broken  off  or  the  glass  needle  of  Wright  may  also  be  used.  To  make  a  glass 
needle,  pull  straight  apart  a  piece  of  capillary  tubing  in  a  very  small  flame.  Tap 
the  fine  point  to  break  off  the  very  delicate  extremity.  Scarcely  any  pain  attends 
the  use  of  such  a  needle.  In  puncturing  either  the  tip  of  the  finger  or  lobe  of  the 
ear  a  quick  piano-touch-like  stroke  should  be  used.  The  ear  is  preferable,  as  it  is 
less  sensitive  and  there  is  less  danger  of  infection.  Before  puncturing,  the  skin 
should  be  cleaned  with  70%  alcohol  and  allowed  to  dry.  It  is  advisable  to  sterilize 
the  needle  before  using  it. 

The  first  drop  of  blood  which  exudes  should  be  taken  up  on  the  paper 
of  the  Tallquist  hsemoglobinometer,  using  subsequent  ones  for  the  blood 
pipettes  and  smears.  If  it  is  necessary  to  make  a  complete  blood  ex- 
amination, it  is  rather  difficult  to  draw  up  the  blood  in  the  pipettes, 


2OO  MICROMETRY   AND   BLOOD   PREPARATIONS 

dilute  it,  and  then  get  material  for  fresh  blood  preparations  and  films 
without  undue  squeezing,  which  is  to  be  avoided.  Of  course,  fresh 
punctures  can  be  made.  Ordinarily,  complete  blood  examinations  are 
not  called  for.  It  is  only  a  white  count  or  a  differential  count  or 
an  examination  for  malaria  that  is  required. 

As  a  practical  point  it  is  very  rare  that  a  red  count  is  indicated.  There  is  one 
point  not  sufficiently  recognized  by  physicians  and  that  is  that  a  routine  blood 
examination  is  not  apt  to  be  as  carefully  conducted  as  one  calling  for  a  specific  fea- 
ture. Without  disparaging  the  necessity  of  routine  examinations  of  urine  as  well 
as  blood  it  is  a  fact  that  the  internist  who  knows  what  he  wants  gets  better  results 
from  the  laboratory  man. 

HEMOGLOBIN  ESTIMATION 

The  most  accurate  instrument  for  this  purpose  is  the  Miescher 
modification  of  the  v.  Fleischl  haemoglobinometer. 

The  magenta-stained  glass  wedge  for  comparison  with  the  diluted  blood  is  similar 
in  each  instrument,  but  by  the  use  of  a  diluting  pipette  accurate  dilutions  are  possible 
in  the  Miescher.  There  are  two  cells  provided — one  12  mm.  high,  the  other  15  mm. 
the  idea  of  this  being  to  enable  one  to  make  separate  comparisons  and  to  select 
the  central  part  of  the  glass-wedge  scale,  where  comparison  is  more  accurate  than 
at  the  ends.  As  these  cells  contain  columns  of  diluted  blood  proportionately  as  5 
to  4,  we  should  have  similar  readings  when  we  multiply  the  reading  on  the  scale 
with  the  15  mm.  cell  by  ££. 

The  mixing  pipette  is  graduated  with  the  marks  J^,  %j  and  K — the  first  giving  a 
dilution  of  i  to  400  (when  the  diluent,  a  0.1%  soda  solution,  is  drawn  up  to  the  mark 
above  the  bulb)  the  second  of  i  to  300  and  the  last  of  i  to  200. 

Artificial  light  preferably  from  a  candle  is  necessary.  There  is  a  table  accom- 
panying each  instrument  which  shows  the  value  for  that  particular  instrument  in 
milligrams  per  liter  of  haemoglobin  for  any  reading  obtained  on  the  scale.  The  nor- 
mal amount  of  haemoglobin  in  the  blood  is  usually  given  as  13  to  14  grams  per  looc.c. 
blood.  For  the  first  two  weeks  after  birth  the  amount  is  much  higher,  16  to  20  grams 
per  100  c.c.  After  this  time  it  begins  to  drop  so  that  a  child  from  five  or  six 
months  to  twelve  or  fifteen  years  old  only  has  about  n  grams  in  100  c.c.  blood. 

Sahli  found  variations  in  normal  individuals  of  from  13.7  grams  to  17.3  grams  per 
100  c.c.  of  blood. 

The  apparatus  is  expensive,  requires  considerable  time  and  care  in  the  making  of 
estimations,  and  is  exclusively  an  instrument  for  a  well-equipped  laboratory. 

Sahli's  Haemometer. — A  simple  and  apparently  very  scientific 
instrument  which  has  been  recently  introduced  is  the  Sahli  modifica- 
tion of  the  Gower  haemoglobinometer.  Instead  of  the  tinted  glass,  or 
gelatin  colored  with  picrocarmine  to  resemble  a  definite  blood  dilution, 
Sahli  uses  as  a  standard  the  same  coloring  matter  as  is  present  in  the 


HAEMOGLOBIN  ESTIMATIONS 


201 


tube  containing  the  blood.  By  acting  on  blood  with  10  times  its 
volume  of  N/io  HC1,  haematin  hydrochlorate  is  produced,  which  gives 
a  brownish-yellow  color.  In  the  standard  tube,  which  is  sealed,  a 
dilution  representing  i%  of  normal  blood  is  used. 

To  apply  this  test,  pour  in  N/io  HC1  to  the  mark  10  on  the  scale  of  the  graduated 
tube.  Add  to  this  20  cu.  mm.  of  the  blood  to  be  examined,  drawn  up  by  the  capil- 
lary pipette  provided.  So  soon  as  the  mixture  as- 
sumes a  clear  bright  dark  brown  color,  add  water 
drop  by  drop  until  the  color  of  the  tubes  matches. 
The  reading  of  the  height  of  the  aqueous  dilution 
on  the  scale  gives  the  Hb.  reading.  The  tubes  are 
encased  in  a  vulcanite  frame  with  rectangular  aper- 
tures. This  gives  the  same  optical  impression  as 
would  planoparallel  glass  sides. 

The  most  accurate  readings  are  obtained  with  arti- 
ficial light  in  a  dark  room  but  almost  as  satisfactory 
comparisons  can  be  obtained  with  natural  light  from 
a  window.  It  is  advisable  to  turn  the  ruled  side 
around  so  that  one  may  match  colors  without  being 
influenced  in  his  determination  by  the  scale. 

The  apparatus  must  be  kept  in  a  dark  place  as 
strong  light  will  change  the  color  of  the  standard 
tube.  It  is  recommended  that  the  N/io  HC1  be 
preserved  with  chloroform. 

Tallquist's  Haemoglobin  Scale. — This  is 
a  small  book  of  specially  prepared  filter- 
paper  with  a  color-scale  plate  of  10  shades 
of  blood  colors.  These  are  so  tinted  as  to 
match  blood  taken  up  on  a  piece  of  the  filter- 
paper  and  are  graded  from  10  to  100.  So 
soon  as  the  blood  on  the  filter-paper  has  lost 
its  humid  gloss,  the  comparison  should  be 
made. 


FIG.     52. — Sahli's     haemo- 
globinometer.     (Greene.) 


This  may  be  done  by  shifting  the  blood-stained  piece  of  filter-paper  suddenly 
from  one  to  the  other  of  the  holes  cut  in  each  shade— the  piece  of  filter-paper  being 
underneath  the  color  plate;  it  is  better,  however,  to  match  the  colors  with  the  blood 
spot  against  the  scale  rather  than  behind  a  preparation.  Grawitz  prefers  to  cut  the 
stained  spot  from  the  filter-paper  and  place  it  directly  on  the  color  scale. 

At  least  a  square  centimeter  of  the  filter-paper  should  be  stained 
by  the  blood.  Daylight  coming  from  a  window  to  the  rear  or  at  the  side 
should  be  used  in  making  the  comparison.  The  error  with  this  method 


2O2 


MICROMETRY   AND  BLOOD   PREPARATIONS 


is  probably  not  over  10%  after  a  little  experience. 
is  not  kept  in  the  dark,  the  tints  tend  to  fade. 


If  the  colored  plate 


To  COUNT  BLOOD-CORPUSCLES 

The  instrument  almost  universally  used  is  the  Thoma-Zeiss  haemacy- 
tometer.  The  apparatus  consists  of  two  pipettes,  one  for  leukocytes, 
graduated  to  give  a  dilution  of  i  to  10  or  greater;  the  other  for  red  cells 
to  give  a  dilution  of  a  i  to  100  or  greater.  The  white  pipette  has  the 
mark  n  above  the  bulb  and  the  red  pipette  the  mark  101.  In  addition, 
there  is  a  counting  chamber. 

This  consists  of  a  square  of  glass  with  a  round  hole  in  the  center.  Occupying  the 
center  of  this  round  hole  is  a  circular  disc  of  glass  of  less  diameter,  so  that  an  encirc- 

ling channel  is  left.  The  square  and  the  circle 
of  glass  are  cemented  to  a  heavy  glass  slide. 
The  surfaces  of  each  are  absolutely  level  and 
highly  polished.  That  of  the  circular  disc  is 
ruled  into  squares  of  varying  size  and  is  exactly 
Ho  rnm.  below  the  level  of  the  surface  of  the  sur- 
rounding glass  square. 


When  a  polished  piano-parallel  cover- 
glass  rests  on  the  shelf,  as  the  outer  square 
glass  is  termed,  there  is  a  space  left  be- 
tween its  under-surface  and  the  ruled  disc 
of  o.i  mm.  The  channel  around  the  disc 
is  termed  the  moat  or  ditch. 

The  most  desirable  rulings  are  those  of  Tiirck 
and  of  Zappert.  In  these  the  entire  ruled  surface 
consists  of  nine  large  squares,  each  i  mm.  square. 
These  are  subdivided,  and  in  the  central  large 
small  squares  used  for  averaging  the  red  cells. 


FIG.  5  3 .  —Thomas-Zeiss 
blood  counter  showing  pipette, 
counting  chamber,  and  ruled 
field.  (Greene.} 


the 


square  are  to  be  found 
These  small  squares  are  Ho  rnm.  square  and  are  arranged  in  nine  groups  of  16 
small  squares  by  bordering  triple-ruled  lines.  As  the  unit  in  blood  counting  is 
the  cubic  millimeter,  if  one  counted  all  the  white  cells  lying  within  one  of  the 
large  squares  (i  mm.  square),  he  would  have  only  counted  the  cells  in  a  layer 
one-tenth  of  the  required  depth,  so  that  it  would  be  necessary  to  multiply  the 
number  obtained  by  10.  This  product,  multiplied  by  the  dilution  of  the  blood, 
would  give  the  number  of  white  cells  in  a  cubic  millimeter  of  undiluted  blood. 
Some  workers  prefer  the  Biirker  hamacytometer.  In  this  there  are  two  ruled 
wedge-shaped  pieces  of  glass,  separated  at  their  bases,  which  take  the  place  of 
the  ruled  disc  of  the  Thoma  apparatus.  Two  oblong  pieces  of  glass  are  on  either 
side  of  the  ruled  wedges  and  are  o.i  mm.  higher,  thus  taking  the  place  of  the  shelf. 
Clamps  fix  a  cover-glass  on  these  shelves  giving  a  space  Ho  mm.  over  the  ruled  sur- 


RED   CELL  COUNTS 


203 


faces.  The  blood  is  run  in  by  capillarity  from  the  mixing  pipette.  I  gave  up  this 
type  of  counter  because  the  clamps  made  manipulation  awkward  and  because  the 
usual  apparatus  is  most  satisfactory. 

Red  Cell  Counts. — To  make  a  red  count:  Having  a  fairly  large  drop  of  blood,  ap- 
ply the  tip  of  the  101  pipette  to  it  and,  holding  the  pipette  horizontally,  carefully 
and  slowly  draw  up  with  suction  on  the  rubber  tube  a  column  of  blood  to  exactly 
0.5  or  i.  The  variation  of  ^5  inch  from  the  mark  would  make  a  difference  of  al- 
most 3%.  If  the  column  goes  above  0.5,  it  can  be  gently  tapped  down  on  a  piece 
of  filter-paper  until  the  0.5  line  is  cut.  Now  insert  the  tip  of  the  pipette  into  some 
diluting  fluid  and,  revolving  the  pipette  on  its  long  axis  while  filling  it  by  suction, 
you  continue  until  the  mark  101  is  reached.  A  variation  of  ^5  inch  at  this  mark 
would  only  give  an  error  of  about  one- thirtieth  of  i%.  After  mixing  thoroughly  by 
shaking  for  one  or  two  minutes,  the  fluid  in  the  pipette  below  the  bulb  is  expelled 
(this,  of  course,  is  only  diluting  fluid).  A  drop  of  the  diluted  blood  of  a  size  just 
sufficient  to  cover  the  disc  when  the  cover-glass  is  adjusted,  is  then  deposited  on  the 
disc  and  the  cover-glass  applied  by  a  sort  of  sliding  movement,  best  obtained  by  using 
a  forceps  in  one  hand  assisted  by  the  thumb  and  index-finger  of  the  other. 

Among  diluting  fluids  Toisson's  is  probably  the  best: 

Sodium  chloride i  gram 

Sodium  sulphate 8  grams 

Glycerine 30  c.c. 

Distilled  water 160  c.c. 

Dissolve  the  sodium  chloride  and  the  sodium  sulphate  in  the  glycerine  water  and 
add  sufficient  methyl  or  gentian  violet  to  give  a  rich  violet  tint. 

Hayem's  solution  is  very  satisfactory  and  is  preferred  by  many  workers.  It  has 
the  following  composition:  Corrosive  sublimate  0.5  gram,  Sod.  chloride  i  gram, 
Sod.  sulphate  5  gram,  200  c.c.  of  distilled  water. 

A  2^%  solution  of  potassium  bichromate  makes  a  very  satisfactory  diluting 
fluid  in  the  counting  of  red  cells. 

A  salt  solution  of  about  i%  strength,  tinged  with  about  i  drop  of  a  saturated 
alcoholic  solution  of  gentian  violet  to  about  50  c.c.,  is  a  good  substitute,  or  the  salt 
solution  alone  will  answer  when  no  white  count  is  to  be  made  at  the  same  time  as  the 
red  one. 

It  is  important  to  work  quickly  in  adjusting  the  cover-glass,  or  there  will  be  cells 
settling  in  the  center  of  the  drop  from  a  greater  depth  than  the  one  which  the  apposi- 
tion of  the  cover-glass  makes  (Ko  mm-  deep). 

A  good  preparation  should  show: 

1.  Presence  of  Newton's  rings. 

2.  Absence  of  air  bubbles. 

3.  Entire  surface  of  ruled  disc  covered. 

4.  Equal  distribution  of  cells. 

Before  counting,  about  five  minutes  should  be  allowed  for  the  set- 
tling of  the  cells. 
It  will  be  remembered  that  the   small   squares   are    ^o    mm.   square.    The 


204  MICROMETRY   AND   BLOOD   PREPARATIONS 


depth  of  fluid  from  upper  surface  of  shelf  to  lower  surface  of  cover-glass  is  Mo  mm. 
Hence  each  space  embraced  by  the  small  square  and  the  depth  of  fluid  is  Mo 00 
of  the  unit  used  in  estimating  number  of  corpuscles  in  blood,  or  i  cu.  mm. 
(Mo  X  Mo  X  Mo  =  Mooo)-  Count  100  of  the  small  squares  (this  enables  one 
to  use  decimals).  There  are  nine  squares  between  triple-ruled  lines,  each  containing 
1 6  small  squares.  Count  the  number  of  corpuscles  in  the  16  small  squares 
contained  in  upper  left-hand  triple-ruled  square.  Put  down  this  count.  Next 
count  corpuscles  in  the  adjoining  1 6  squares.  Put  down  this  count.  Then  in 
third  1 6  squares.  Put  down  the  number.  Now  move  down  to  next  row  of 
three  triple-ruled  squares.  Count  the  number  of  corpuscles  in  each  of  the  three 
i6-square  spaces  and  set  down  the  numbers  for  addition.  We  have  now  counted 
96  small  squares  (6  X  16).  Count  at  any  place  four  additional  small  squares  and 
add  number  of  blood-cells  contained  therein  to  those  in  the  96  small  squares 
already  counted.  Divide  the  sum  by  100  or  simply  point  off  two  decimals. 
This  gives  the  average  for  each  small  square.  Multiply  this  by  the  dilution  and  then 
(as  the  small  square  is  only  Mooo  cu.  mm.)  by  4000.  This  will  give  the  number  of 
corpuscles  in  i  cu.  mm.  Example:  100  small  squares  contained  655  red  cells. 
Pointing  off,  6.55  equals  average  number  of  red  cells  per  small  square.  Multiply 
by  dilution  (200)  and  then  by  4000  (the  small  square  is  4000  times  smaller  than  the 
unit:  i  cu.  mm.) — 6.55  X  200  =  1310  X  4000  =  5,240,000. 

At  least  ioo  small  squares,  and  preferably  200  should  be  counted. 
If  the  blood  appears  normal,  one  may  simply  count  the  number  of  red 
cells  in  five  of  the  16  small  square  spaces  (80  small  squares).  Having 
added  the  numbers  and  multiplying  by  10,000,  you  obtain  the  number 
of  cells  in  i  cu.  mm.  (Eighty  small  squares  is  J-^Q  of  the  unit  of  i 
cu.  mm.,  or  4000  small  squares.  The  blood  dilution  being  i  to  200,  we 
have  50  X  200  X  number  of  cells  in  80  small  squares.) 

In  counting,  count  corpuscles  lying  on  the  lines  above  and  to  the  right.  Do  not 
count  those  lying  on  lines  below  and  to  the  left. 

In  the  small  squares  count  only  corpuscles  lying  in  the  space  or  cutting  the  upper 
line.  This  prevents  counting  the  same  cell  twice. 

To  Count  White  Cells. — Draw  up  the  blood  in  the  white  pipette  to 
the  mark  0.5.  Then,  still  holding  the  pipette  as  near  the  horizontal  as 
possible,  because  the  column  of  blood  tends  to  fall  down  in  the  larger 
bore,  draw  up  by  suction  a  diluting  fluid  which  will  disintegrate  the  red 
cells  without  injuring  the  whites. 

The  best  fluid  is  0.5%  of  glacial  acetic  acid  in  water.  This  makes  the  white  cells 
stand  out  as  highly  refractile  bodies.  Some  prefer  to  tinge  the  fluid  with  gentian 
violet.  The  0.5  mark  is  preferred  because  it  takes  a  very  large  drop  of  blood  to 
fill  the  tube  up  to  the  i  mark  and  if  there  is  much  of  a  leukocytosis  a  i  to  10  dilution 
is  not  sufficient.  In  leukaemic  blood  it  is  better  to  use  the  red  pipette  with  the 
0.5%  acetic  acid  solution. 

The  blood  having  been  drawn  up  to  0.5,  we  have  a  dilution  of  i  to  20. 


LEUCOCYTE  COUNTS  205 

Making  a  preparation,  exactly  as  was  done  in  the  case  of  the  red  count,  we  count 
all  of  the  white  cells  in  one  of  the  large  squares  (i  sq.  mm.).  The  cross  ruling  greatly 
facilitates  this.  Note  the  number.  Then  count  a  second  and  a  third  large  square. 
Strike  an  average  for  the  large  squares  counted  and  multiply  this  by  10,  as  the  depth 
of  the  fluid  gives  a  content  equal  to  only  ^ 0  cu.  mm.  Then  multiply  by  the 
dilution.  Example:  First  large  square  50;  second  large  square  70;  third  large 
square  60.  Average  60.  Then  60  X  10  X  20  =  12,000,  the  number  of  leukocytes  in 
i  cu.  mm.  of  blood.  The  count  may  be  made  with  a  low  power  (%-inch  objec- 
tive) as  the  leukocytes  stand  out  like  pearls.  It  is  more  accurate,  however,  to  use  a 
higher  power,  so  that  pieces  of  foreign  material  may  be  recognized  and  not  enumer- 
ated as  white  cells.  If  one  will  accustom  himself  to  comparing  the  distribution  of 
the  leukocytes  in  a  well-made  stained  dried  blood  film,  prepared  according  to  Ehrlich's 
cover-glass  method,  with  that  in  a  haemacytometer  preparation,  he  can  readily 
acquire  an  experience  which  will  enable  him  to  determine  with  considerable  accuracy 
the  degree  of  leukocytosis  by  the  examination  of  a  stained  cover-glass  preparation 
alone.  Furthermore,  one  can  identify  the  leukocytes  in  a  Giemsa-stained  smear 
with  the  %-inch  objective.  This  is  specially  true  of  the  large  mononuclears  and 
transitionals  whose  increase  has  such  significance  in  the  tropics. 

Special  diaphragms  for  the  ocular,  with  a  square  opening,  which  just  covers  one 
of  the  large  squares  of  the  haemacytometer  (400  small  squares)  may  be  purchased 
or  one  may  cut  a  square  hole  from  a  round  piece  of  stiff  paper  which  rests  on  the  ocular 
micrometer  supports.  This  is  easily  measured  by  noting  the  number  of  spaces  on 
the  ocular  micrometer  which  equals  one  side  of  the  square.  With  such  an  opening 
one  can  count  the  leukocytes  in  unruled  areas  equal  to  a  large  square,  when  the  ruling 
is  less  convenient  than  is  present  in  the  Ttirck  ruling. 

Combined  Red  and  White  Counts. — In  the  absence  of  a  white  pipette  or  when 
it  is  desired  to  make  a  white  count  with  the  same  preparation  as  is  used  for  the  red 
one,  especially  if  the  ruling  is  of  the  old  style  (only  central  ruling  and  not  in  nine 
large  squares  as  with  Zappert  and  Tiirck),  it  is  advisable  to  make  use  of  the  method 
of  counting  by  fields.  With  a  Leitz  No.  4  ocular  and  a  No.  6  objective,  with  a  tube 
length  of  120  mm.,  it  will  be  observed  that  the  field  so  obtained  has  a  diameter 
of  eight  small  squares.  Now,  remembering  that  the  area  of  a  circle  equals  the  square 
of  the  radius  multiplied  by  TT,  or  3.1416,  we  have  the  following  calculation:  The 
diameter  being  eight  small  squares,  the  radius  would  be  four  small  squares.  Squar- 
ing the  radius,  we  have  16.  This  multiplied  by  3.1416  gives  us  50.  This 
means  that  every  field,  with  the  microscope  adjusted  as  stated,  contains  50  of 
the  small  squares,  or  Ho  of  the  unit  of  i  cu.  mm.  of  the  diluted  blood. 

By  keeping  a  single  red  cell  in  view  while  moving  the  mechanical  stage  from  right 
to  left  or  from  above  downward,  we  know  that  a  new  field  of  50  small  squares  is 
brought  into  view  when  the  circumference  of  the  field  cuts  this  individual  cell. 
Example:  As  2000  small  squares  would  ordinarily  be  a  sufl&cient  number  to  count 
for  a  white  count,  this  would  require  us  to  count  the  number  of  leukocytes  in  40 
of  the  designated  microscopic  fields  (this,  of  course,  is  only  one-half  the  unit,  hence 
we  should  multiply  by  2).  Counted  40  fields  and  noted  50  white  cells.  50  X  2 
=  100  X  200  (the  dilution  in  red  pipette)  =  20,000.  Consequently  20,000  would 
represent  the  number  of  leukocytes  in  i  cu.  mm.  of  the  blood  examined. 

Cleaning  Apparatus.— After  making  a  blood  count,  the  haemacytometer  slide 
should  be  cleaned  with  soap  and  water  and  then  rubbed  dry,  preferably  with  an  old 


206  MICROMETRY   AND  BLOOD   PREPARATIONS 

piece  of  linen.  As  the  accuracy  of  the  counting  chamber  depends  upon  the  integrity 
of  the  cement,  any  reagent  such  as  alcohol,  xylol,  etc.,  and,  in  particular,  heat,  will 
ruin  the  instrument.  The  pipettes  should  be  cleaned  by  inserting  the  ends  into  the 
tube  from  a  vacuum  pump,  as  a  Chapman  pump.  First  draw  water  or  i%  sod. 
carbonate  solution  through  the  pipette,  then  alcohol,  then  ether,  and  finally  allow 
air  to  pass  through  to  dry  the  interior.  If  the  interior  is  stained,  use  i%  HC1  in 
alcohol.  If  a  vacuum  pump  is  not  at  hand,  a  bicycle  pump  or  suction  by  mouth 
will  answer. 

PREPARATIONS  FOR  THE  STUDY  OF  FRESH  BLOOD 

Many  authorities  prefer  a  fresh-blood  specimen  to  a  stained  dried 
smear  in  the  study  of  parasites  of  the  blood.  In  malaria  in  particular 
there  is  so  much  information  as  to  species  to  be  obtained  from  a  fresh 
specimen  that  the  employment  of  this  method  should  never  be  neglected. 
While  waiting  for  the  film  to  stain  one  has  five  or  six  minutes  which 
could  not  be  better  spent  than  in  examining  the  fresh  specimen  which 
only  requires  a  moment  to  make. 

Manson's  Method.— Have  a  perfectly  clean  cover-glass  and  slide.  Touch  the 
apex  of  the  exuding  drop  of  blood  with  the  cover-glass  and  drop  it  on  the  center  of 
the  slide.  The  blood  flows  out  in  a  film  which  exhibits  an  "empty  zone"  in  the 
center.  Surrounding  this  we  have  the  "zone  of  scattered  corpuscles,"  next  the 
"single  layer  zone"  and  the  "zone  of  rouleaux"  at  the  periphery.  It  is  well  to  ring 
the  preparation  with  vaseline.  When  desiring  to  demonstrate  the  flagellated  bodies 
in  malaria,  it  is  well  to  breathe  on  the  cover-glass  just  prior  to  touching  the  drop  of 
blood. 

The  Method  of  Ross  is  very  easy  of  application  and  gives  most  satisfactory 
preparations.  Take  a  perfectly  clean  slide,  and  make  a  vaseline  ring  or  square  of 
the  size  of  the  cover-glass.  Then,  having  taken  up  the  blood  on  the  cover-glass, 
drop  it  so  that  its  margin  rests  on  the  vaseline  ring.  Gently  pressing  down  the  cover- 
glass  on  the  vaseline  makes  beautiful  preparations  which  keep  for  a  very  long  time. 
If  it  is  desired  to  study  the  action  of  stains  on  living  cells,  this  method  is  also  appli- 
cable. A  very  practical  way  to  do  this  is  to  tinge  0.85%  salt  solution  containing  i% 
sodium  citrate  (the  same  as  is  used  in  opsonic  work)  with  methylene  azur,  gentian 
violet,  or  methyl  green.  With  a  Wright  bulb  pipette,  take  up  i  part  of  blood, 
then  i  part  of  tinted  salt  solution.  Mix  them  quickly  on  a  slide  and  then  deposit 
a  small  drop  of  the  mixture  in  the  center  of  the  vaseline  ring  and  immediately  apply 
a  cover-glass  and  press  down  the  margins  as  before.  This  method  will  be  found  of 
great  practical  value. 

A  METHOD  FOR  MAKING  DIFFERENTIAL  LEUKOCYTE  COUNT  IN  SAME 
PREPARATION  AS  FOR  WHITE  COUNT 

Employ  the  same  technic  as  in  making  the  ordinary  white  count 
but  using  as  a  diluting  fluid  a  ij^  or  2%  formalin  solution  to  which  has 


DIFFERENTIAL  COUNTS  207 

been  added  i  drop  of  Giemsa's  stain  for  each  c.c.  just  before  making 
the  blood  examination. 

The  best  results  are  obtained  when  the  mixing  in  the  pipette  bulb  is  done  im- 
mediately after  taking  up  the  blood  and  diluent.  Recently  I  have  found  it  necessary 
to  add  enough  N/i  NaOH  to  the  commercial  formalin  to  bring  it  to  about  +0.75. 
To  do  this  add  to  it  a  few  drops  of  phenolphthalein  as  an  indicator  and  continue 
to  add  a  dilute  sodium  hydrate  or  sodium  carbonate  solution  until  a  pink  color 
just  developes  at  room  temperature.  This  corresponds  to  about  +0.75  with  boiling 
titration.  The  acidity  of  commercial  "Formalins"  varies  greatly.  Of  this  +0.75 
formalin  I  use  i^%  in  a  %%  glycerine  solution  instead  of  water. 

The  usual  technic  in  making  the  haemocytometer  preparation  is  employed  using 
a  Tiirck  ruling.  Count  the  leukocytes  in  the  three  upper  or  lower  i  sq.  mm.  squares, 
divide  by  3  to  obtain  an  average  per  sq.  mm.,  multiply  by  10  for  the  content  of  a 
cubic  millimeter  and  then  by  20  for  the  dilution.  (Blood  to  0.5;  diluent  to  n.) 
This  can  be  done  mentally  and  requires  no  calculation  on  paper.  Having  counted 
the  leukocytes,  again  go  over  the  same  portion  of  the  ruled  surface  and  count  the 
polymorphonuclears  and  estimate  the  percentage  of  these  to  the  total  leukocytes. 
The  majority  of  disrupted  cells  in  a  dry-stained  preparation  are  transitionals  hence 
the  percentage  of  polymorphonuclears  by  this  method  is  lower. 

It  is  unnecessary  in  such  a  count  to  have  an  assistant;  of  course,  in  making  a 
complete  differential  count  it  is  preferable  to  have  some  one  tabulate  or  laboriously 
to  do  this  one's  self. 

The  red  cells  are  practically  diaphanous  and  not  disintegrated  as 
when  acetic  acid  is  used  as  a  diluent,  consequently  it  is  easy  to  make 
out  the  particular  red  cell  as  to  size,  etc.,  containing  a  malarial  parasite. 

The  best  results  are  obtained  with  a  %-inch  objective.  Higher  powers  are  of 
course  impracticable  by  reason  of  the  thickness  of  the  cover-glass  of  the  haemocyto- 
meter. 

The  following  are  the  appearances  of  the  various  leukocytes. 

Eosinophiles. — In  these  the  bilobed  nucleus  stains  rather  faintly  and  the  color 
is  greenish  blue.  The  eosinophile  granules  show  easily  as  coarse,  brickdust-red 
colored  particles. 

Polymorphonuclears. — The  nucleus  stains  a  deep,  rich,  pure  violet  but  less  in- 
tense than  that  of  the  small  lymphocyte.  The  shape  of  the  nucleus  is  typically 
three  or  four  lobed  but  even  when  of  the  horseshoe  shape  of  a  transitional  nucleus 
is  easily  recognizable  by  the  intensity  of  the  violet  staining.  That  which  makes  the 
polymorphonuclears  very  easy  of  differentiation  is  the  distinctness  of  the  cell  out- 
lines produced  by  the  fine  yellowish  granulations  in  the  cytoplasm. 

Small  Lymphocytes. — The  nucleus  is  perfectly  round  and  stains  a  deep  violet. 
It  is  almost  impossible  to  make  out  any  cytoplasmic  fringe. 

Large  Lymphocytes. — The  nucleus  here  is  round  and  of  a  lighter  violet  than  that 
of  the  small  lymphocyte.  The  cytoplasm  is  blue,  nongranular,  and  sharply  defined 
from  the  nucleus. 

Large  Mononuclears. — These  show  a  washed-out,  slate-colored  nucleus  which 
blends  with  the  gray  slate-blue  staining  of  the  cytoplasm  so  that  there  is  an  in- 
defmiteness  of  outline  in  the  more  or  less  irregularly  contoured  nucleus. 


208  MICROMETRY   AND   BLOOD   PREPARATIONS 

Transitionals. — These  show  the  same  characteristics  as  the  large  mononuclears, 
but  with  a  more  faintly  stained  and  more  indented  nucleus.  The  large  mono- 
nuclears and  transitionals  stand  out  as  slate-colored  cells.  When  very  much  degen- 
erated these  cells  have  a  greenish  hue. 

Mast  Cells. — The  granulations  show  as  a  rich  maroon  or  reddish-violet  color 

The  young  ring  forms  of  malaria  show  as  violet-blue  areas  in  the  red  cells.  When 
half-grown  or  approaching  the  merocyte  stage,  the  containing  red  cell  takes  on  a 
faint  pink  coloration,  thereby  differentiating  it  from  the  noninfected  red  cells.  At 
the  same  time  the  parasite  is  extruded  and  has  the  appearance  of  a  violet-blue  body 
projecting  from  the  margin  of  the  red  cell.  It  is  as  if  a  blue  body  were  budding  from 
a  pink  one.  Crescents  stand  out  very  distinctly. 

It  is  an  easy  matter  with  this  method  to  count  the  number  of  trypanosomes  or 
malarial  crescents  in  a  cubic  millimeter  of  blood. 


PREPARATIONS  AND  STAINING  OF  DRIED  FILMS 

When  preparations  are  desired  for  a  differential  count,  Ehrlich's 
method  of  making  films  is  to  be  preferred,  as  the  different  types  of 
leukocytes  are  more  evenly  distributed.  In  making  smears  by  spread- 
ing, there  is  a  tendency  for  the  polymorphonuclears  to  be  concentrated 
at  the  margin  while  lymphocytes  remain  in  the  central  part  of  the  film. 

In  Ehrlich's  method  we  have  perfectly  clean  dry  cover-slips.  Take  up  a  small 
drop  of  blood  without  touching  the  surface  of  the  ear  or  finger.  Drop  this  cover- 
glass  immediately  on  a  second  one  and  as  soon  as  the  blood  runs  out  in  a  film,  draw 
the  two  cover-slips  apart  in  a  plane  parallel  to  the  cover-glasses.  Slide  them  apart. 
Ehrlich  uses  forceps  to  hold  the  cover-glasses  to  avoid  moisture  from  the  fingers, 
but  I  find  I  can  work  more  quickly  and  satisfactorily  with  the  fingers  alone.  In 
making  malarial  smears  it  is  better  to  wash  the  finger  or  ear  with  soap  and  water 
to  get  rid  of  all  grease  and  dirt.  Then  dry  thoroughly  before  puncturing.  Alcohol 
is  not  so  efficient. 

Slides  and  spreaders  should  be  absolutely  clean  and  grease  free.  Scrubbing  with 
soap  and  water,  thorough  rinsing  and  drying,  then  subjecting  the  slide  to  the  flame 
to  make  it  grease  free  is  satisfactory. 

Of  the  various  methods  of  spreading  films  on  slides  there  is  none 
equal  to  that  described  by  Daniels.  In  this  the  drop  of  blood  is  drawn 
along  and  not  pushed  along.  The  films  are  even,  can  be  made  of  any 
desired  thickness  by  changing  the  angle  of  the  drawing  slide,  and  there 
is  little  liability  of  crushing  pathological  cells.  Take  a  small  drop  of 
blood  on  the  end  of  a  clean  slide.  Touch  a  second  slide  about  % 
inch  from  end  with  the  drop  and  as  soon  as  the  blood  runs  out  along 
the  line  of  the  slide  end,  slide  it  at  an  angle  of  45°  to  the  other  end  of  the 
horizontal  slide.  The  blood  is  pulled  or  drawn  behind  the  advancing 


BLOOD   SMEARS 


2OO 


edge  of  the  advancing  slide.     An  angle  less  than  45°  makes  a  thinner 
film;  one  greater,  a  thicker  film. 

Instead  of  a  slide  a  square  cover-glass  may  be  used  and  if  the  edge  be  smooth  it 
makes  a  more  satisfactory  spreader  than  the  slide. 

Instead  of  the  Daniels  method  some  prefer  to  take  up  the  drop  of  blood  on  the 
slide  on  which  the  smear  is  to  be  made,  about  Y2  inch  from  the  end.  Then  apply 
the  spreader  slide  and  so  soon  as  the  drop  runs  along  the  end  of  the  spreader  slide 
proceed  as  above  described. 


FIG.  54. — Blood  technic.  i,  2,  3,  Method  for  making  blood  smear  on  slide;  4, 
U  tube  for  resting  slides  while  staining;  5,  slide  showing  grease  pencil  marking, 
marking  prevents  stain  from  overflowing;  6,  method  for  drawing  apart  cover-glasses 
in  making  blood  smear. 

Of  the  various  methods  of  making  smears  by  means  of  cigarette  paper,  rubber 
tissue,  needles,  etc.,  the  best  seems  to  be  to  take  a  piece  of  capillary  glass  tubing  and 
use  this  instead  of  a  needle  in  making  the  film.  There  is  one  advantage  about  the 
strip  of  cigarette  paper  touched  to  the  drop  of  blood  and  drawn  out  along  the  slide 
or  cover-glass,  and  that  is  that  it  is  almost  impossible  not  to  make  a  working  prepara- 
tion by  this  method. 

In  the  making  of  smears  the  chief  points  are  to  make  the  smears  as 
soon  after  taking  the  blood  as  possible  and  to  have  slides  and  cover- 
glasses  scrupulously  clean.     It  is  well  to  flame  all  slides  and  cover- 
14 


210  MICROMETRY   AND   BLOOD   PREPARATIONS 

glasses  which  are  to  be  used  for  blood-work.     This  is  the  best  method 
of  getting  rid  of  grease. 

Thick  Film  Methods. — Such  methods  are  of  the  greatest  practical 
value  in  diagnosis  of  malarial  parasites  when  in  very  small  numbers 
in  the  peripheral  circulation.  They  are  also  of  great  value  in  finding 
trypanosomes,  relapsing  fever  spirochaetes  and  filarial  embryos  in  the 
blood.  Ruge's  method  so  brings  out  the  polymorphonuclears  that 
such  a  technic  can  be  used  for  opsonic  index. 

Method  of  Ross. — In  this  about  one-half  of  a  drop  of  blood  is  smeared  out  over  a 
surface  about  equal  to  that  of  a  square  cover-glass  and  allowed  to  dry.  It  is  then 
flooded  with  a  0.1%  aqueous  solution  of  eosin  for  about  fifteen  minutes.  The  prepa- 
ration is  then  gently  washed  with  water  and  then  treated  with  a  polychrome 
methylene-blue  solution.  After  a  few  seconds  this  is  carefully  washed  off  and  the 
preparation  dried  and  examined. 

Method  of  James. — -James  smears  out  an  ordinary  drop  of  blood  so  that  it  makes  a 
circular  smear  about  %  inch  in  diameter.  This  may  be  easily  accomplished  with  a 
spatulate  wooden  toothpick.  When  dry,  treat  the  blood  smear  with  alcohol  contain- 
ing HC1.  (Alcohol  50  c.c.  HC1.  10  drops)  until  the  haemoglobin  is  dissolved  out. 
Then  wash  thoroughly  in  water  for  five  or  ten  minutes.  Allow  to  dry  and  then 
stain  as  ordinarily  with  the  Wright  or  Giemsa  stain. 

Ruge's  Method. — The  best  thick  film  method  is  that  of  Ruge.  After  the  blood 
has  dried  well,  gently  move  the  slide  about  in  a  glass  containing  a  2%  solution  of 
formalin  to  which  has  been  added  i%  of  glacial  acetic  acid.  After  laking  is  com- 
plete, as  shown  by  disappearance  of  brown  color,  treat  the  slide  in  the  same  way  in  a 
glass  of  tap  water  to  remove  all  traces  of  acid.  Next  wash  very  gently  in  distilled 
water  and  stain  with  dilute  Giemsa  (i  drop  to  i  c.c.)  for  twenty  to  thirty  minutes. 
Wash  in  water  and  allow  to  dry  without  heat  or  blotting  paper. 

Some  workers  prefer  to  stain  the  dried  thick  smear  for  one  hour  in  a  jar  containing 
dilute  Giemsa  stain  (i  to  40)  without  previous  fixation  or  dehaemoglobinization. 

At  present,  I  make  my  thick  films  by  taking  up  a  moderately  large 
loopful  from  the  exuding  drop  of  the  puncture  wound. 

This  is  deposited  at  one  end  of  the  slide  and  from  it  three  or  four  more 
daubs  are  made  in  succession  toward  the  other  end  of  the  slide. 
These  daubs  are  quickly  smeared  out  before  coagulation  takes  place 
in  the  first  daub. 

With  all  thick  film  methods  it  is  extremely  important  to  have  thorough  drying 
of  the  smear  before  dehaemoglobinizing  or  staining.  This  ordinarily  requires  one  or 
two  hours  in  the  air  or  twenty  to  thirty  minutes  in  the  incubator.  It  is  particularly 
important  in  working  with  such  smears,  although  holding  for  ordinary  smears,  to 
protect  them  from  flies,  ants,  etc.,  as  such  insects  will  eat  up  the  smear  in  a  few 
minutes  if  left  exposed. 

Fixation  of  Film. — In  Wright's,  Leishman's,  and  other  similar  stains  the  methyl- 
alcohol  solvent  causes  the  fixation.  In  staining  with  Giemsa's  stain,  Ehrlich's 


STAINING   BLOOD-FILMS  211 

triacid,  hasmatoxylin  and  eosin,  Smith's  formol  fuchsin,  and  with  thionin,  separate 
fixation  is  necessary.  For  Giemsa  and  thionin,  either  absolute  alcohol  (ten  to  fifteen 
minutes),  or  methyl  alcohol  (two  to  five  minutes)  answer  well. 

Formalin  vapor,  for  five  to  ten  seconds,  is  also  used  for  fixation.  For  Ehrlich's 
triacid,  haematoxylin  and  eosin  and  formol  fuchsin,  heat  gives  the  best  results. 
The  best  method  is  to  place  the  films  in  an  oven  provided  with  a  thermometer. 
Raise  the  temperature  of  the  oven  to  i35°C.  and  then  remove  the  burner.  After 
the  oven  has  cooled,  take  out  the  fixed  slides  or  slips. 

Some  prefer  to  place  a  crystal  of  urea  on  the  slide,  then  hold  it  over  the  flame 
until  the  urea  melts.  This  shows  that  a  temperature  between  130°  and  i35°C.  has 
been  reached. 

One  of  the  handiest  methods  is  to  drop  a  few  drops  of  95%  alcohol  on  the  slide 
or  cover-glass.  Allow  this  to  flow  over  the  entire  surface;  then  get  rid  of  the  exeess 
of  alcohol  by  touching  the  edge  to  a  piece  of  filter-paper  for  a  second  or  two.  Then 
light  the  remaining  alcohol  film  from  the  flame  and  allow  the  burning  alcohol  to 
burn  itself  out.  A  chemical  fixation  which  gives  good  fixation  for  haematoxylin 
and  triacid  stains  (not  equal  to  heat)  is  a  modification  of  Zenker's  fluid  (Whitney). 
To  Miiller's  fluid,  which  is  potassium  bichromate  2  grams,  sodium  sulphate  i  gram, 
and  water  100  c.c.,  add  5  grams  of  bichloride  of  mercury  and  5  c.c.  of  nitric  acid 
(C.  P.).  Fixation  is  obtained  in  five  seconds. 

When  using  corrosive  sublimate  fixation  one  should  after  thorough  washing  in 
water  treat  the  film  with  Gram's  iodine  solution  for  about  two  minutes  and  then  wash 
with  70%  alcohol  until  the  yellow  color  of  the  film  disappears.  Methyl  alcohol  for 
two  minutes  is  satisfactory.  (See  Staining  of  Protozoa.) 

Staining  Blood-films. — As  separate  staining  with  eosin  and  methyl- 
ene  blue  rarely  gives  good  preparations  and  as  the  modifications  of  the 
Romanowsky  stain  recommended  are  easy  to  make  and  employ,  and 
give  much  greater  information,  the  separate  method  of  staining  is  not 
recommended.  The  most  satisfactory  single  stain  is  thionin. 

Rees'  Thionin  Solution— Take  of  thionin  1.5  grams,  alcohol  10  c.c.,  aqueous  solution 
of  carbolic  acid  (5%)  100  c.c.  Keep  this  as  a  stock  solution.  It  should  be  at  least 
two  weeks  old  before  using.  For  use,  filter  off  5  c.c.  and  make  up  to  20  c.c.  with 
water. 

1.  Fix  films  (a)  by  heat,  (b)  by  alcohol  and   ether,  or  (c)  preferably  by    i% 
formalin  in  95%  alcohol  for  one  minute. 

2.  Stain  for  from  ten  to  twenty  minutes.     Wash  and  mount.     Malarial  parasites 
are  stained  purplish;  nuclei  of  leukocytes,  blue;  red  ceUs,  faint  greenish  blue. 

Ehrlich's  Triacid  or  Triple  Stain.— There  are  required: 

1.  Sat.  aq.  sol.  orange  G.     (Dissolve  3  grams  in  50  c.c.  water.) 

2.  Sat.  aq.  sol.  acid  fuchsin.     (Dissolve  10  grams  in  50  c.c.  water.) 

3.  Sat.  aq.  sol.  methyl  green.     (Dissolve  10  grams  in  50  c.c.  water.) 

These  three  solutions  may  be  kept  as  stock  solutions.  They  keep  well  in  the  dark. 
To  make  the  stain,  add  9  c.c.  of  No.  2  (acid  fuchsin)  to  18  c.c.  of  No.  i  (orange  G.). 
After  they  are  mixed  thoroughly,  add  20  c.c.  of  No.  3  (methyl  green).  Then  after 
the  first  three  ingredients  are  well  mixed,  add  5  c.c.  of  glycerine.  Mix,  then  add  15 


212  MICROMETRY  AND  BLOOD  PREPARATIONS 

c.c.  of  alcohol;  again  mix,  and  finally  add  30  c.c.  of  distilled  water.  Keep  the 
mixed  stain  about  one  week  before  using.  The  best  fixatives  are  heat  and  Whitney's 
modified  Zenker.  To  use,  stain  films  from  two  to  five  minutes;  then  wash  and 
mount.  The  triacid  stain  is  a  good  tissue  stain.  The  objections  to  the  triacid 
stain  are  that  it  does  not  stain  malarial  parasites  or  mast  cells  and  that  failure  to 
obtain  good  results  is  of  frequent  occurrence. 

Wright's  Method. — The  stain  is  made  by  adding  i  gram  of  methylene  blue  (Grub- 
ler)  to  100  c.c.  of  a  %%  solution  of  sodium  bicarbonate  in  water.  This  mixture  is 
heated  for  one  hour  in  an  Arnold  sterilizer.  The  flask  containing  the  alkaline  methyl- 
ene-blue  solution  should  be  of  such  size  and  shape  that  the  depth  of  the  fluid  does 
not  exceed  2%  inches.  When  cool,  add  to  the  methylene-blue  solution  500  c.c.  of 
a  i  to  1000  eosin  solution  (yellow  eosin,  water  soluble).  Add  the  eosin  solution 
slowly,  stirring  constantly  until  the  blue  color  is  lost  and  the  mixture  becomes 
purple  with  a  yellow  metallic  luster  on  the  surface,  and  there  is  formed  a  finely 
granular  black  precipitate.  Collect  this  precipitate  on  a  filter-paper  and  when 
thoroughly  dry  (dry  in  the  incubator  at  38°C.)  dissolve  0.3  gram  in  100  c.c.  of  pure 
methyl  alcohol  (acetone  free).  Wright  lately  has  recommended  using  o.i  in  60  c.c. 
methyl  alcohol.  This  constitutes  the  stock  solution.  For  use  filter  off  20  c.c.  and 
add  to  the  filtrate  5  c.c.  of  methyl  alcohol. 

A  modification  by  Batch  is  very  satisfactory.  In  this  method  instead  of  poly- 
chroming  the  methylene  blue  with  sodium  bicarbonate  and  heat,  the  method  of 
Borrel  is  used.  Dissolve  i  gram  of  methylene  blue  in  100  c.c.  of  distilled  water. 
Next  dissolve  0.5  gram  of  silver  nitrate  in  50  c.c.  of  distilled  water.  To  the  silver 
solution  add  a  2  to  5%  caustic  soda  solution  until  the  silver  oxide  is  completely  pre- 
cipitated. Wash  the  precipitated  silver  oxide  several  times  with  distilled  water. 
This  is  best  accomplished  by  pouring  the  wash-water  on  the  heavy  black  precipitate 
in  the  flask,  agitating,  then  decanting  and  again  pouring  on  water.  After  removing 
all  excess  of  alkali  by  repeated  washings,  add  the  methylene-blue  solution  to  the 
precipitated  silver  oxide  in  the  flask.  Allow  to  stand  about  ten  days,  occasionally 
shaking  until  a  purplish  color  develops.  The  process  may  be  hastened  in  an  incu- 
bator. When  polychroming  is  complete,  filter  off  and  add  to  the  filtrate  the  i  to 
1000  eosin  solution  and  proceed  exactly  as  with  Wright's  stain. 

In  Leishman's  method  the  polychroming  is  accomplished  by  adding  i  gram  of 
methylene  blue  to  100  c.c.  of  a  V^%  solution  of  sodium  carbonate.  This  is  kept  at 
65°C.  for  twelve  hours  and  allowed  to  stand  at  room  temperature  for  ten  days 
before  the  eosin  solution  is  added.  The  succeeding  steps  are  as  for  Wright's  stain. 

In  all  Romanowsky  methods  distilled  water  should  be  used.  If  not 
obtainable,  the  best  substitute  is  rain-water  collected  in  the  open  and 
not  from  a  roof. 

If  the  yellow  color  in  water  in  a  test-tube  to  which  has  been  added  a  small  pinch  of 
haematoxylin  does  not  change  to  blue  in  from  one  to  five  minutes  it  is  too  acid  and 
should  be  treated  with  a  i  %  sod.  carb.  sol.  until  it  does  show  blue.  Alkaline  waters 
are  less  easy  to  correct. 

Method  of  staining: 

1.  Make  films  and  air  dry. 

2.  Cover  dry  film  preparation  with  the  methyl-alcohol  stain  for  one  minute  (to 
fix). 


GIEMSA'S  STAIN  213 

3.  Add  water  to  the  stain  on  the  cover-glass  or  slide,  drop  by  drop,  until  a  yellow 
metallic  scum  begins  to  form.     It  is  advisable  to  add  the  drops  of  water  rapidly  in 
order  to  eliminate  precipitates  on  the  stained  film.     Practically,  we  may  add  i  drop  of 
water  for  every  drop  of  stain  used. 

4.  Wash  thoroughly  in  water  until  the  film  has  a  pinkish  tint. 

5.  Dry  with  filter-paper  and  mount.     The  stained  preparation  is  less  apt  to  show 
foreign  material  and  damage  if  one  allows  the  film  to  dry  without  blotting.     In  a 
moist  atmosphere  one  may  dry  the  film  high  over  the  flame  but  any  near  contact 
with  the  heat  of  the  flame  is  injurious. 

Red  cells  are  stained  orange  to  pink;  nuclei,  shades  of  violet;  eosino- 
phile  granules,  red;  neutrophile  granules,  yellow  to  lilac;  blood  platelets, 
purplish;  malarial  parasites,  blue;  chromatin,  metallic  red  to  rose  pink. 

The  bottle  in  which  the  methyl  alcohol  solution  is  kept  must  be  tightly  stoppered 
with  a  cork  stopper  and  kept  in  the  dark.  Any  evaporation  of  the  alcohol  interferes 
with  proper  fixation  of  the  blood  film  and  the  light  affects  the  delicate  stain. 

Giemsa's  Modification  of  the  Romanowsky  Method. — This  is  one  of 
the  most  perfect  of  the  modifications.  The  objection  is  that  greater 
time  in  staining  films  is  required  than  with  the  Wright  or  Leishman 
method  and  the  stain  is  very  expensive. 

Take  of  Azur  II  eosin  0.3  gram.     Azur  II  0.08  gram. 

Dissolve  this  amount  of  dry  powder  in  25  c.c.  of  pure  anhydrous  glycerine  at  6o°C. 
Then  add  25  c.c.  of  methyl-alcohol  at  the  same  temperature.  Allow  the  glycerine 
methyl-alcohol  solution  to  stand  over  night  and  then  filter.  This  is  the  stock  stain. 
To  use:  Dilute  i  c.c.  with  10  to  15  c.c.  of  distilled  water.  If  i  to  1000  potassium 
carbonate  solution  is  used  instead  of  water  it  stains  more  deeply. 

The  alkaline  diluent  is  used  to  obtain  the  course  stippling  in  malig- 
nant tertian  (Maurer's  clefts).  Having  fixed  the  smear  with  methyl 
alcohol  for  one  to  five  minutes,  pour  on  the  diluted  stain,  and  after 
fifteen  to  thirty  minutes  wash  off  and  continue  washing  with  distilled 
water  until  the  film  has  a  slight  pink  tinge.  For  Treponema  pallidum 
stain  from  two  to  twelve  hours. 

While  the  Romanowsky  methods  are  more  satisfactory  for  differential  counts  and 
for  the  demonstration  of  the  malarial  parasites,  and  especially  for  differentiating 
species,  yet  by  reason  of  the  liability  to  deterioration  in  the  tropics  of  methylene 
blue  the  haematoxylin  methods  may  be  preferable.  Many  workers  in  blood-work 
and  cytodiagnosis  prefer  the  haematoxylin. 

1.  Fix  the  film  either  by  heat,  with  methyl  alcohol  for  two  minutes  or  with  Whit- 
ney's fixative.     Heat  is  to  be  preferred. 

2.  Stain  with  Meyer's  hemalum  or  Delafield's  haematoxylin  for  from  five  to 
fifteen  minutes  according  to  the  stain.     Frequently  three  minutes  will  be  found 
sufficient.     To  make  the  hemalum,  dissolve  0.5  gram  of  haematin  in  25  c.c.  of  95% 


214  MICROMETRY  AND  BLOOD   PREPARATIONS 


,. 

tis- 


alcohol.     Next  dissolve  25  grams  of  ammonia  alum  in  500  c.c.  of  distilled  wat 
Mix  the  two  solutions  and  allow  to  ripen  for  a  few  days.     The  stain  should  be  satis- 
factory in  two  or  three  days. 

To  make  Dela field's  haematoxylin,  dissolve  i  gram  of  haematoxylin  crystals  in 
6  c.c.  of  95%  alcohol.  Add  this  to  100  c.c.  of  saturated  aqueous  solution  of  ammonia 
alum.  After  exposure  to  light  for  a  week,  the  color  changes  to  a  deep  blue  purple. 
Add  to  this  ripened  stain  25  c.c.  of  glycerine  and  25  c.c.  of  methyl-alcohol  and,  after 
it  has  stood  for  about  two  days,  filter.  The  stain  should  be  filtered  from  time  to  time 
as  a  sediment  forms.  This  makes  a  stock  solution  which  should  be  diluted  10  to  15 
times  with  water  when  staining. 

Mink's  Modification  of  Unna's  Haematoxylin 

Haematoxylin i  gram 

Alum 8  grams. 

Sulphur  (sublimed) i  gram. 

Glycerine 30  c.c. 

Alcohol 50  c.c. 

Water 100  c.c. 

Dissolve  the  haematoxylin  in  the  glycerine  in  a  mortar.  Dissolve  the  alum  in  the 
water  and  add  it  to  the  glycerine  hsematoxylin  in  the  mortar.  Then  add  the  sulphur 
and  the  alcohol.  The  solution  ripens  in  about  three  to  four  days.  Allow  the  sedi- 
ment to  remain  in  the  bottom  of  the  bottle  containing  the  stain  and  filter  off  small 
quantities  as  needed. 

3.  Wash  for  two  to  five  minutes  in  tap  water  to  develop  the  haematoxylin  color. 

4.  Stain  either  with  a  i  to  1000  aqueous  solution  of  eosin  or  with  a  one-half  of  i  % 
eosin  solution  in  70%  alcohol.     The  eosin  staining  only  requires  fifteen  to  thirty 
seconds. 

5.  Wash  and  examine. 

IODOPH3LIA 

This  reaction  is  supposed  to  be  due  to  the  presence  of  glycogen, 
especially  in  the  polymorphonuclears,  in  suppurative  conditions. 

It  has  been  stated  that  a  differentiation  between  the  joint  involvement  in  gonor- 
rhceal  infection  and  in  articular  rheumatism  may  be  made  from  iodophilia  being 
present  in  the  gonococcus  infection. 

Make  blood-smears  on  cover-glasses  as  usual,  and  after  they  dry,  but  without 
fixation,  mount  them  in  a  drop  of  the  following  solution: 

Iodine i  part. 

Potassium  iodide 3  parts. 

Gum  arabic 50  parts. 

Water 100  parts. 

Small  brown  masses  in  the  polymorphonuclears  indicate  a  positive  iodophilia. 
Viscosity  of  the  Blood. — This  is  estimated  by  observing  the  relative  height  to  which 
blood  rises  in  capillary  tubes  as  compared  with  water,  and  normally  varies  from  three 


COAGULATION  RATE  215 

to  five.     The  higher  the  haemoglobin  content  the  greater  the  viscosity.     Viscosity  is 
high  in  arterio-sclerosis  and  diabetic  coma,  low  in  the  anaemias  of  nephritis. 

Coagulation  Rate  of  Blood. — This  determination  is  of  value  in 
connection  with  operations  on  jaundiced  patients. 

Wright's  coagulometer  is  a  standard  instrument  but  is  cumbersome. 

A  simple  method  of  determining  the  rate  is  to  take  a  piece  of  capillary  glass  tubing 
and  hold  it  downward  from  the  puncture  to  let  it  fill  for  3  or  4  inches.  Then  at  inter- 
vals of  thirty  seconds  scratch  with  a  file  the  capillary  tubing  at  short  distances  and 
break  off  between  the  fingers.  When  coagulation  has  taken  place  a  long  worm-like 
coagulum  is  obtained.  Normally  coagulation  occurs  in  about  three  to  four  minutes, 
when  the  temperature  is  that  of  the  hand  in  which  the  tubes  are  conveniently  held. 
Rudolf  recommends  placing  the  tubes  in  metal  tube  containers  in  a  Thermos  bottle 
at  2o°C.  He  gives  the  normal  coagulation  rate  for  this  temperature  as  eight  minutes, 
while  at  a  temperature  below  this  the  period  is  lengthened.  Age  and  sex  do  not 
influence  the  rate.  Sabrazes,  the  originator  of  this  method  found  no  appreciable 
variation  in  tubes  from  0.8  to  1.2  mm.  diameter. 

In  Barker's  test  you  mix  a  drop  of  blood  and  a  drop  of  distilled  water  on  a  slide 
and  with  a  capillary  tube  sealed  off  at  the  end  stir  the  mixture  every  half  minute. 
So  soon  as  fibrin  threads  appear  you  have  coagulation. 

SPECIFIC  GRAVITY  OF  THE  BLOOD 

Hammerschlag  has  a  method  for  the  determination  of  the  Hb. 
percentage  based  upon  the  specific  gravity  of  the  blood. 

In  this  method  a  mixture  of  benzol  and  chloroform  is  made  of  a  specific  gravity  of 
about  1050.  A  medium  size  drop  of  blood  is  then  taken  up  with  a  pipette  and  intro- 
duced below  the  surface  of  the  mixture,  carefully  avoiding  production  of  bubbles. 
Variation  in  temperature  introduces  a  very  appreciable  error.  If  it  sinks  add  more 
chloroform  from  a  dropping  bottle,  if  it  tends  to  rise,  more  benzol.  The  mixture  in 
which  the  drop  of  blood  tends  to  remain  stationary,  near  the  top  of  the  mixed  benzol 
and  chloroform,  has  the  same  specific  gravity  as  that  of  the  blood.  This  is  deter- 
mined by  an  accurately  graduated  hydrometer.  The  normal  average  specific 
gravity  for  men  is  1059,  for  women  1056.  A  table,  giving  the  Hb.  percentage  cor- 
responding to  the  specific  gravity,  accompanies  the  outfit. 

Eykmann  controls  the  specific  gravity  of  the  drop  of  blood  by  adding  a  drop  c 
salt  solution  made  to  have  a  similar  specific  gravity. 

The  specific  gravity  is  reduced  in  all  anaemias,  especially  chlorosis;  in  nephritis  witl 
osdema  as  well  as  in  most  cachetic  states. 

In  these  latter  the  Hb.  percentage  may  be  normal. 

Specific  Gravity  in  Cholera.— To  determine  the  necessity  for  intraven- 
ous infusion  in  cholera  Rogers  has  recently  recommended  the  employ- 
ment of  small  bottles  containing  aqueous  solution  of  glycerine  with 
specific  gravities  varying  from  1048^0  1070,  increasing  the  spec 
gravity  in  each  successive  bottle  by  2°. 


2l6  MICROMETRY   AND  BLOOD    PREPARATIONS 

An  accurate  urinometer  will  suffice  to  determine  the  specific  gravity.  Drops  of 
blood  from  the  cholera  patient  are  deposited  at  the  center  of  the  surface  of  the  fluid 
in  the  bottles  from  a  capillary  pipette.  If  the  specific  gravity  of  the  blood  is  1062 
at  least  a  liter  of  saline  or  sodium  bicarbonate  solution  is  needed.  If  1066  at  least 
2  liters.  Formerly  he  estimated  the  indications  by  blood-pressure  considering  a 
pressure  of  80  in  Europeans  or  of  70  in  natives  as  indicating  intravenous  injections. 

TESTS  FOR  AGGLUTINATION  AND  HAEMOLYSIS  OF  THE  RED  CELLS 
(TRANSFUSION) 

In  the  selection  of  a  donor  for  blood  for  transfusion  it  is  always 
necessary  to  try  his  red  cells  against  the  serum  of  the  recipient  as  well 
as  the  patient's  red  cells  against  the  serum  of  the  donor,  in  order  to 
prove  the  absence  of  haemolyzing  or  agglutinating  bodies. 

Certain  persons  have  isohaemolysins  in  their  blood  which  dissolve  the  red  cells  of 
other  persons  and  in  paroxysmal  haemoglobinuria  autohaemolysins  may  be  present 
which  can  destroy  the  patient's  own  red  cells.  This  autohaemolysin  seems  operative 
only  when  a  low  temperature  is  followed  by  a  high  one.  When  haemoglobinaemia 
exists  the  liver  converts  it  into  bile  pigment,  causing  bilious  stools  and  jaundice.  If 
one-sixth  of  the  red  cells  are  destroyed  hEemoglobinuria  results. 

Before  transfusing  carry  out  the  following  tests: 

From  a  vein  take  about  i  c.c.  of  blood  in  a  centrifuge  tube  containing  i  %  of  sod. 
citrate  salt  solution;  then  shift  the  stopper  of  the  blood  system  to  a  dry  centrifuge 
tube  and  draw  into  it  about  3  or  4  c.c.  of  blood.  Throw  down  the  citrated  blood, 
pipette  off  the  supernatant  fluid  and  wash  the  sediment  with  normal  saline. 

Again  pipette  off  the  saline  after  centrifuging  and  make  a  10%  emulsion  of  the  red- 
cell  sediment  in  normal  saline. 

Centrifuge  the  coagulated  blood  in  the  other  tube  and  collect  the  serum  which 
separates  from  the  clot. 

Carry  out  these  procedures  for  both  donor  and  recipient. 

Tests:  i.  In  a  small  test-tube  deposit  i  drop  of  the  donor's  10%  red-cell  emulsion 
and  then  add  4  drops  of  the  recipient's  serum. 

2.  Treat  similarly  i  drop  of  the  recipient's  red-cell  emulsion  with  4  drops  of  the 
donor's  serum. 

3.  Treat  i  drop  of  donor's  red-cell  emulsion  with  4  drops  of  his  serum. 

4.  Treat  i  drop  of  recipient's  red-cell  emulsion  with  4  drops  of  his  serum.     Finally 
add  i  c.c.  of  salt  solution  to  each  of  the  four  tubes,  shake  gently  and  place  in  incubator 
for  two  hours. 

Tests  3  and  4  should  fail  to  show  either  agglutination  or  haemolysins. 
Some  prefer  to  keep  the  tubes  over  night  in  ice-box  after  the  preliminary  examina- 
tion following  incubation. 

OCCULT  BLOOD 

When  the  presence  of  blood  cannot  be  recognized  by  macroscopical 
or  microscopical  methods  (occult  blood)  we  must  resort  to  spectro- 


OCCULT  BLOOD  TESTS 


2I7 


scopic  or  chemical  tests.  It  is  in  connection  with  blood  in  the  faces 
that  these  tests  for  occult  blood  are  chiefly  called  for.  Before  making 
such  tests  on  faeces  it  is  advisable  to  have  the  patient  on  a  meat-free 
and  green-vegetable-free  diet  for  two  or  three  days.  It  is  chiefly  in 
carcinoma  or  ulcerations  of  the  gastro-intestinal  tract  that  such  exami- 
nations of  the  faeces  are  required. 

Haemin  Crystal  Test  (Teichmann).— Prepare  a  solution  of  o.i  gram  each  of  KI, 
KBr,  and  KCL  in  100  c.c.  of  acetic  acid.  This  is  a  stable  solution.  Mix  some  of 
the  material  with  a  few  drops  of  the  solution  on  a  slide,  apply  a  cover-glass  and 
warm  the  material  until  bubbles  begin  to  appear  (gentle  steaming),  then  examine  for 
dark-brown  crystals. 

Blood  in  the  Urine. — The  most  rapid  method  of  detection  is  by  using  the  micro- 
spectroscope.  An  ordinary  hand  spectroscope  will  answer  however. 

Donogany's  test  is  very  satisfactory.  To  10  c.c.  of  urine  add  i  c.c.  ammonium 
sulphide  solution  and  i  c.c.  of  pyridin.  The  urine  will  assume  a  more  or  less  deep 
orange  color  according  to  its  blood  content.  The  spectrum  of  alkaline  methajmo- 
globin  or  haemochromogen  will  be  obtained.  See  illustrations  under  urine. 

In  making  the  guaiac  or  other  tests  it  is  a  good  plan  to  repeatedly  filter  the  blood- 
containing  urine  through  the  filter.  Then  touch  a  spot  on  the  moist  filter  with  the 
guaiac  or  benzidin  solution  and  then  finally  drop  on  this  so  treated  spot  a  drop  or 
two  of  hydrogen  peroxide  solution. 

Blood  in  Faeces  or  Gastric  Contents.— Take  5  grams  of  faeces  and  rub  it  up 
thoroughly  in  a  mortar  with  15  c.c.  of  a  mixture  of  equal  parts  of  alcohol,  glacial 
acetic  acid  and  ether.  Filter  through  an  unmoistened  pleated  filter-paper  re- 
peatedly until  only  3  to  4  c.c.  remain  of  the  filtrate.  The  fasces  filtrate  can  be 
first  tested  chemically  by  depositing  a  few  drops  in  the  center  of  three  or  four 
circles  of  white  filter-paper  placed  in  a  Petri  dish  or  upon  an  ordinary  white  plate. 

Weber  Guaiac  Test. — The  moistened  spot  is  then  treated  with  a  few  drops  of  a 
freshly  prepared  alcoholic  solution  of  guaiac  resin  (about  3^  gram  of  guaiac  resin  is 
broken  up  into  small  fragments  and  shaken  up  in  about  3  c.c.  of  alcohol)  and  finally 
there  is  dropped  upon  the  spot  a  few  drops  of  a  solution  of  hydrogen  peroxide. 
Waves  of  blue  color  extending  out  into  the  moistened  filter-paper  show  a  positive 
test  for  blood. 

Benzidin  Test. — For  the  benzidin  test  pour  on  this  faeces  filtrate-moistened  filter- 
paper  a  few  drops  of  the  following  solution:  2  c.c.  of  a  saturated  alcoholic  solution  of 
benzidin  2  c.c.  of  solution  of  peroxide  of  hydrogen  and  2  drops  of  glacial  acetic  acid. 
(Blue.) 

If  the  aloin  test  is  preferred  we  treat  the  filtrate-moistened  filter-paper  with  a 
few  drops  of  a  3%  solution  of  aloin  in  70%  alcohol  and  then  treating  the  spot  with 
hydrogen  peroxide  solution.  Brick-red  color. 

Phenolphthalin  Test. — The  phenolphthalin  test  is  an  extremely  delicate  one  and 
will  show  the  pink  color  at  times  with  certain  specimens  of  water,  hence  one  should 
always  make  a  control  using  the  reagents  without  addition  of  the  suspected  blood 
material. 

In  my  opinion  it  has  great  value  as  a  negative  test. 

Take  2  c.c.  of  the  ether  alcohol  acetic-acid  filtrate  and  dilute  with  7  or  8  c.c.  water. 


2l8  MICROMETRY  AND  BLOOD   PREPARATIONS 

Neutralize  the  acidity  with  sodium  hydrate.  Then  add  i  c.c.  of  the  phenolphthalin 
reagent,  mix  and  finally  add  several  drops  of  i  to  10  dilution  of  peroxide  of  hydrogen 
and  note  the  formation  of  a  decided  rose-pink  coloration,  varying  in  depth  according 
to  the  amount  of  occult  blood. 

To  prepare  the  reagent  dissolve  i  or  2  grams  phenolphthalein  and  25  grams  KOH  in 
100  c.c.  distilled  water.  Add  10  grams  powdered  zinc  and  heat  gently  until  solution 
is  decolorized.  Phenolphthalin  is  a  reduction  product  of  phenolphthalein. 

More  Reliable  is  the  Spectroscopic  Test. — For  this  we  take  about  3  c.c. 
of  the  concentrated  ether,  acetic  acid,  alcohol  faecal  nitrate  and  add  to  it 
2  c.c.  of  pyridin.  Then  add  not  more  than  2  to  3  drops  of  ammonium 
sulphide  solution.  (The  ammonium  sulphide  solution  should  be  kept 
in  an  amber-colored,  glass-stoppered  bottle.  The  solution  should  be 
freshly  prepared  every  ten  days.)  Examine  the  solution,  contained  hi  a 
small  test-tube,  with  the  spectroscope  and  the  two  absorption  bands  of 
methaemoglobin-alkaline  (haemochromogen),  between  D  and  E,  show 
a  positive  blood  test.  Comparison  should  be  made  with  fresh  blood, 
in  which  the  absorption  band  in  the  yellow  is  nearer  line  D  (oxy- 
haemoglobin  spectrum). 

The  great  trouble  about  the  spectroscopic  test  is  that  it  will  only  show  the  presence 
of  quite  large  amounts  of  blood.  //  is  by  no  means  a  delicate  test. 

ACIDOSIS  AND  METHODS  FOR  ITS  DETERMINATION 

Everyone  is  familar  with  that  form  of  respiratory  disturbance 
associated  with  diabetic  coma,  known  as  Kussmaul's  air  hunger.  Here 
we  have  hyperpnoea,  a  form  of  dyspnoea  typically  without  cyanosis, 
and  the  best  clinical  evidence  of  acidosis. 

Reduced  alkalinity  of  the  blood  would  be  a  better  expression  than  acidosis  because 
even  a  neutral  reaction  of  the  blood  would  be  incompatible  with  maintenance  of  life. 

Acidosis  is  an  important  consideration  in  alimentary  tract  disturb- 
ances of  infants  and  children,  as  in  infantile  diarrhoeas  or  cyclical 
vomiting  of  children.  It  is  not  infrequent  in  the  pneumonias  of 
children.  In  adults  we  must  keep  the  possibility  of  the  occurrence  of 
acidosis  in  mind  in  the  vomiting  and  eclampsia  of  pregnancy,  in  sal- 
icylate  poisoning,  following  chloroform  anaesthesia,  and  in  chronic 
nephritis  as  well  as  in  many  infectious  diseases. 

Sellard's  alkaline  treatment  for  the  acidosis  of  cholera  is  a  measure  of  the  utmost 
value. 

Starvation,  whether  the  result  of  gastric  ulcer,  gastric  carcinoma  or  otherwise,  is  a 
recognized  cause  of  acidosis.  The  insistence  upon  one-sided  diets  in  children  is  often 


ACIDOSIS  219 

the  cause  of  increased  acid  content  of  the  blood  and  such  causes  are  not  absent  in 
many  of  the  dietary  treatments  of  diseases  of  adults,  whether  in  the  height  of  the 
disease  or  during  convalescence. 

From  the  laboratory  standpoint  acidosis  is  usually  recognized  by  an  increase  in  the 
urinary  acetone  bodies  or  by  noting  an  increase  in  the  ammonia  quotient  due  to 
demands  upon  the  alkali  for  neutralization  of  the  increased  acid  production.  Ace- 
tone, diacetic  acid  and  /3-oxybutyric  acid  (acetone  bodies)  come  from  abnormal  met- 
abolism of  fats.  When  not  formed  in  excessive  amounts  the  two  acids  are  con- 
verted into  acetone  and  appear  in  the  urine  as  such.  With  more  marked  production 
acetone  formation  falls  behind  and  diacetic  acid  appears  or,  with  still  more  marked 
acid  production,  /3-oxybutyric  acid. 

These  acids,  of  themselves,  seem  harmless  and  their  injurious 
action  is  connected  with  abstraction  of  alkali  from  the  blood. 

There  are  some  who  think  lactic  acid  may  be  formed  from  abnormal  metabolism 
of  carbohydrates  and  that  certain  cases  of  acidosis  failing  to  show  increase  of  acetone 
bodies  may  be  connected  with  lactic  acid  increase.  Of  course,  retention  of  acids  of 
normal  metabolism  would  also  cause  acidosis.  To  prevent  this  the  organism 
utilizes  a  sufficient  amount  of  the  ammonia  from  protein  or  rather  amino-acid 
metabolism  instead  of  further  changing  it  into  urea.  So  that  an  increased  am- 
monia quotient  may  signify  that  nature  has  control  of  the  abnormal  acid  production. 

In  the  acidosis  connected  with  chronic  nephritis  the  preformed 
ammonia  may  be  lower  than  normal  indicating  some  defect  in  the 
mechanism  of  ammonia  neutralization  of  acids. 

Acetone  bodies  can  come  from  proteins  as  well  as  fats.  Whatever  may  be  the 
explanation  of  abnormal  formation  of  acetone  bodies  it  seems  to  be  associated  with 
inability  of  the  organism  to  obtain  sugar  for  its  tissues,  hence  the  therapeutic  value 
of  giving  glucose  in  acidosis  conditions  where  the  trouble  is  carbohydrate  deficiency. 
Glucose  administration  is  frequently  combined  with  alkali  treatment  in  acidosis. 
Of  course,  where  the  trouble  is  an  inability  on  the  part  of  the  cells  to  utilize  the  sugar, 
which  may  be  circulating  in  greatly  increased  amounts  in  the  blood,  from  lack  of 
pancreatic  internal  secretion  (as  in  diabetes),  sugar  injection  would  have  no  effect  on 
this  abnormal  acid  production. 

Even  in  ordinary  metabolism  great  amounts  of  acid  are  produced 
but  these  are  eliminated  normally  by  way  of  lungs  as  well  as  urine. 
In  this  connection  a  failure  on  the  part  of  the  kidneys  to  remove 
acid  would  result  in  acid  retention  and,  if  sufficiently  marked,  acidosis. 

Besides  the  phosphoric  and  sulphuric  acid  produced  in  the  metabolism  of  the 
phosphorus  and  sulphur  of  proteids  we  have  enormous  amounts  of  carbonic  acid 
formed  in  the  tissues. 

The  alkalinity  of  the  blood  is  maintained  by  the  bicarbonate  of  soda, 
by  sodium  and  potassium  phosphates  and  to  some  extent  proteins  can 


220  MICROMETRY   AND   BLOOD   PREPARATIONS 


. 


neutralize  acids.  The  carbonic  acid  is  taken  up  by  the  bicarbonate 
of  the  blood  and  gotten  rid  of  as  CO2  by  the  lungs,  without  any  loss  of 
sodium  bicarbonate. 

Other  metabolic  acids,  however,  cause  a  loss  of  sodium  bicarbonate  (and  along 
with  this  the  other  blood  alkalis)  so  that  the  determination  of  the  lowering  of  this 
salt  in  the  blood  indicates  an  acidosis.  This  may  be  carried  out  by  van  Slyke's 
method  described  below. 

It  has  been  found  that  the  alveolar  CO2  falls  with  a  fall  in  the  plasma 
bicarbonate.  (This  however  does  not  hold  with  cardio-respiratory 
cases.)  Therefore  by  determining  the  CO2  content  of  the  expired  air 
upon  forced  expiration  we  can  judge  as  to  reduction  of  blood  carbonate 
and  of  course  acidosis. 

Sellards'  method  for  determination  of  serum  acidosis  is  quite  simple  and  reliable. 
To  carry  out  the  test  we  add  i  c.c.  of  serum,  drop  by  drop,  to  25  c.c.  of  absolute 
alcohol  (it  is  very  important  to  secure  a  neutral  alcohol  and  I  have  found  that  of 
Merck  satisfactory).  This  precipitates  the  protein  which  is  the  factor  interfering 
with  a  sharp  end  reaction.  After  filtering  we  add  3  or  4  drops  of  neutralized  phe- 
nolphthalein  solution  to  the  fitrate  and  evaporate  the  alcohol  in  a  porcelain  dish  on 
a  water-bath.  Every  piece  of  apparatus  must  be  perfectly  dry  and  the  steam  vapor 
of  the  bath  quite  low  to  avoid  the  taking  up  by  the  alcohol  of  water.  In  normal 
cases  the  dark  pinkish  tinge  of  the  sediment,  after  evaporation,  will  remain  at  least 
one  hour,  while  with  cases  showing  increased  acidosis  the  reddish  tinging  of  the 
sediment  disappears  in  a  few  minutes.  An  electric  bath  is  desirable. 

A  very  simple  way  of  determining  bicarbonate  diminution  is  the 
test  for  tolerance  of  alkalis.  The  giving  of  5  grams  of  bicarbonate  of 
soda  to  a  normal  person  on  a  mixed  diet  will  bring  about  an  alkaline 
urine.  Boiling  the  urine  will  bring  out  the  alkalinity  to  litmus  more 
sharply. 

These  amounts  are  increased  until  the  urine  becomes  alkaline.  In  some  cases  of 
acidosis  massive  doses  of  bicarbonate,  as  150  grams  in  one  or  two  days  fail  to  produce 
an  alkaline  urine. 

Plasma  Bicarbonate. — Van  Slyke  has  devised  an  apparatus  for  the  determination 
of  the  carbon  dioxide  capacity  of  oxalate  plasma. 

He  uses  i  c.c.  of  the  plasma  which  is  shaken  with  air  containing  6%  of  CO2  and 
is  introduced  into  the  following  apparatus.  A  50  c.c.  pipette-shaped  apparatus  is 
provided  at  top  and  bottom  with  three-way  stopcocks  and  connected  with  a  bulb  of 
mercury.  The  pipette  is  first  filled  with  mercury  and  the  plasma  having  been  intro- 
duced followed  by  i  c.c.  of  water  and  0.5  c.c.  of  N/i  acid  the  mercury  is  withdrawn 
from  the  pipette  by  lowering  the  bulb  reservoir,  thus  creating  a  Torricellian  vacuum. 
The  CO2  escapes  from  the  solution  as  the  result  of  a  few  minutes  shaking  and  the 
watery  solution  is  drawn  off  from  the  lower  cock.  The  mercury  is  again  made  to 
fill  the  pipette  by  the  other  entrance  of  the  three-way  cock  at  the  bottom  and  rises 


ACIDOSIS   TESTS  221 

to  the  level  of  the  CO2.     This  volume  is  read  off  in  the  calibrated  upper  stem  of 
the  pipette  (calibrated  in  0.02  c.c.  divisions)  and  the  calculations  made  accordingly. 
Normal  serum  binds  about  75%  of  its  volume  of  CO2  while  in  acidosis  figures  as 
low  as  20%  may  be  obtained. 

For  the  determination  of  the  CO2  content  of  alveolar  air  the  apparatus 
of  Fridericia  is  quite  practical.  In  this  the  volume  of  CO?  in  100  c.c. 
of  alveolar  air  is  obtained.  In  such  methods  temperature  plays  so 
important  a  part  in  estimating  volume  that  it  is  hardly  applicable 
except  in  the  hands  of  one  accustomed  to  gas  analysis. 

We  have  constructed  an  apparatus,  using  a  Sedgwick  aerobioscope  as  an  air 
chamber  and  fitting  the  ends  with  glass  stopcocks.  The  patient  having  expired, 
to  get  rid  of  air  in  upper  air  passages,  then  with  forced  expiration  fills  the  200  c.c. 
chamber  of  the  aerobioscope,  immediately  afterward  closing  the  stopcocks.  We 
then  introduce  5  c.c.  of  N/i  KOH,  shaking  at  intervals  for  one-half  hour  to  allow 
absorption  of  CO2  by  the  KOH.  We  then  add  a  few  drops  of  phenolphthalein  as 
indicator  and  titrate  in  the  aerobioscope  chamber  the  loss  in  alkalinity  of  the 
KOH,  using  N/i  HC1.  (For  hydrogen-ion  concentration  method  see  Appendix.) 

Besides  the  tests  for  serum  acidosis  of  Sellards  and  that  for  carbon 
dioxide  content  of  alveolar  air  we  should  also  determine  ammonia 
nitrogen  output  as  well  as  its  quotient. 

Urinary  tests  for  acidity  and  acetone  bodies  content  of  the  urine  should  also  be 
made  as  well  as  that  for  alkali  tolerance.  These  tests  are  all  very  simple  and  can  be 
easily  carried  out  by  any  well-trained  laboratory  worker. 


[AFTER  XIV 
NORMAL  AND  PATHOLOGICAL  BLOOD 

IN  considering  what  may  be  termed  normal  blood,  it  must  be  borne 
in  mind  that  the  normal  varies  for  men,  women,  and  children: 

Hb.  Red  cells  Leukocytes 

Men,  90  to  110%,     5  to  5^  million,        7500 

Women,       80  to  100%,     4}^  to  5  million,        7500 
Children,      70  to  80%,       4^  to  5  million,        9000 

COLOR  INDEX 

This  is  obtained  by  dividing  the  percentage  of  the  haemoglobin  by 
the  percentage  of  red  cells,  5,000,000  red  cells  being  considered  as 
100%. 

To  obtain  the  percentage  of  red  cells  it  is  only  necessary  to  multiply  the  two  ex- 
treme figures  to  the  left  by  two.  Thus  if  a  count  showed  the  presence  of  1,700,000 
red  cells,  the  percentage  would  be  34(17  X  2  =  34).  If  the  Hb.  percentage  in  this 
case  were  50;  then  the  color  index  would  be  50  -5-  34,  or  1.4. 

In  normal  blood  the  color  index  is,  approximately,  i. 

In  anaemias  we  have  three  types  of  color  index:  i.  The  pernicious  anaemia  type, 
which  is  above  i.  Here  we  have  a  greater  reduction  in  red  cells  than  we  have  of  the 
haemoglobin  content  of  each  cell.  2.  The  normal  type,  when  both  red  cells  and 
haemoglobin  are  proportionally  decreased,  as  in  anaemia  following  haemorrhage. 
3.  The  chlorotic  type.  Here  there  is  a  great  decrease  in  haemoglobin  percentage, 
but  only  a  moderate  decrease  in  the  number  of  red  cells.  Hence  the  color  index 
is  only  a  fraction  of  i.  For  example,  in  a  case  of  chlorosis  we  have  40%  of  haemo- 
globin and  90%  of  red  cells,  40  -f-  90  =  0.4. 

RED  CELLS 

In  considering  the  corpuscular  richness  of  a  specimen  of  blood,  it 
must  be  remembered  that  this  does  not  necessarily  bear  any  relation  to 
the  quantity  of  blood  in  the  body.  Thus,  a  more  or  less  bloodless- 
looking  individual,  the  total  quantity  of  whose  blood  is  greatly  reduced, 

222 


PATHOLOGICAL  RED  CELLS  223 

may,  notwithstanding,  give  a  normal  red  count.     In  examining  a  speci- 
men of  peripheral  blood  we  get  a  qualitative,  not  a  quantitative  result. 

Normally,  we  have  an  increase  in  red  cells  in  those  living  at  high  altitudes.  An 
altitude  of  2000  feet  may  increase  the  red  count  about  1,000,000,  and  a  height 
of  6000  feet  about  2,000,000.  Profuse  sweats  and  diarrhoeas  also  increase  the 
red  count.  Pathologically,  in  chronic  polycythemia  with  cyanosis  and  splenic 
enlargement,  we  have  a  red  count  of  about  10,000,000.  In  cyanosis  from  heart 
disease,  etc.,  and  in  Addison's  disease  there  is  also  an  increase  in  red  cells. 

The  normal  red  cell  or  erythrocyte  measures  about  7.5/1  in  diameter.  It  is  non- 
nucleated  and  normally  stains  with  acid  dyes,  taking  the  pink  of  eosin  or  the  orange 
of  orange  G.  If  larger,  10  to  20/1,  it  is  called  a  macrocyte;  if  smaller,  3  to  6/i,  a 
microcyte. 

Anisocytosis  is  a  term  applied  to  a  condition  where  marked  varia- 
tion in  size  of  the  red  cells  occurs. 

Macrocytes  are  rather  indicative  of  severe  forms  of  anaemia,  the  microcytes,  of 
less  grave  types.  When  the  red  cell  is  distorted  in  shape,  it  is  called  a  poikilocyte. 
Care  must  be  exercised  that  distorted  shapes  are  not  due  to  faulty  technic.  Crena- 
tion  and  vacuolation  of  red  cells  are  marked  in  poorly  prepared  specimens. 

In  addition  to  variation  in  size  and  shape,  we  also  have  pathological  variation  in 
staining  affinities. 

Achromia. — This  is  characterized  by  pallor  of  the  central  portion  of 
the  stained  red  cell.  It  also  shows  as  a  central  vacuolation  in  fresh 
blood  and  is  apt  to  deceive  one  in  the  anaemic  blood  of  malaria. 

Polychromatophilia.— This  shows  itself  by  red  cells  taking  a  brownish 
to  a  dirty  blue  tint,  as  is  frequently  seen  in  immature  red  cells,  especially 
nucleated  ones. 

Granular  basophilic  degeneration  (also  termed  punctate  baso- 
philia  and  stippling)  refers  to  the  presence  of  blue  dots  in  the  pink  back- 
ground of  stained  red  cells.  It  is  found  in  many  severe  anaemias,  as 
pernicious  anaemia,  the  leukaemias,  malarial  cachexia,  etc.  It  is  very 
characteristic  of  lead  poisoning. 

The  nucleated  red  cell,  while  normal  for  the  marrow,  is  always 
pathological  for  the  blood  of  the  peripheral  circulation. 

Normoblasts  have  the  diameter  of  a  normal  red  cell.  The  nucleus  is  round  and 
stains  intensely  with  basic  dyes,  often  appearing  almost  black.  Another  character- 
istic is  that  it  frequently  appears  as  does  the  setting  in  a  ring.  Some  give  the  term 
microblast  to  smaller  nucleated  forms.  In  normoblasts  the  red  cell  proper  stains 
normally.  The  Wgaloblasts  not  only  have  a  greater  diameter  than  the  normoblast, 
but  the  nucleuses  poor  in  chromatin,  stains  less  intensely  and  is  less  distinctly  out- 
lined. Instead  of  being  round,  the  nucleus  is  irregular  and  may  be  trefoil  in  shape. 
The  cytoplasm  surrounding  the  nucleus  shows  polychromatophilia.  This  con- 
trasted with  the  pure  blue  of  the  lymphocytes  should  differentiate. 


224  NORMAL   AND   PATHOLOGICAL  BLOOD 


Normoblasts  are  found  in  secondary  anaemias,  and  especially  in 
myelogenous  leukaemia.  Megaloblasts  are  peculiarly  characteristic  of 
pernicious  anaemia.  Enormous  megaloblasts  are  sometimes  termed 
gigantoblasts. 

In  aplastic  anaemia  (  a  severe  type  of  pernicious  anaemia),  in  contrast  to  ordinary 
pernicious  anaemia,  nucleated  reds  are  very  rarely  found.  There  is  also  very  little 
poikilocytosis,  and  the  color  index  is  about  normal.  It  is  a  rare,  rapidly  fatal 
anaemia,  particularly  of  young  women. 

It  does  not  show  remissions,  runs  a  rapid  course,  and  is  attended  with  a  marked 
increase  of  lymphocytes.  The  bone  marrow  of  the  femur  is  pinkish  yellow  and 
homogeneous. 

The  term  leukancemia  has  been  employed  to  describe  conditions 
which  partake  of  the  characteristics  of  pernicious  anaemia  and  leukaemia. 

WHITE  CELLS 

Owing  to  the  conflicting  views  as  to  origin,  nature,  and  functions  of 
the  various  leukocytes,  their  classification  is  in  a  state  of  confusion. 

As  regards  the  appearance  of  the  cells,  this  of  course  varies  as  the  stain  used,  and 
it  requires  considerable  experience  for  a  single  individual  to  be  able  to  positively 
recognize  the  difference  between  a  lymphocyte  and  a  large  mononuclear  when  one 
specimen  is  stained  with  a  Romanowsky  stain,  another  with  Ehrlich's  triacid,  and  a 
third  with  a  haematoxylin  and  eosin.  This,  of  course,  is  intensified  when  different 
persons  adhere  to  the  method  of  staining  which  they  prefer  and  are  at  a  loss  to 
appreciate  differences  which  are  brought  out  by  some  other  stain  used  by  some  other 
person.  Even  with  the  same  stain  used  with  different  specimens  of  blood  we  find  the 
staining  characteristics  of  various  leukocytes  imperceptibly  merging  the  one  into 
the  other,  so  that  at  times  it  is  impossible  for  one,  even  with  his  own  standard  of 
differentiation,  to  be  sure  whether  he  is  dealing  with  a  lymphocyte  or  a  large  mononu- 
clear. The  difficulty  is  even  greater  when  we  deal  with  Tiirck's  irritation  forms  and 
with  myelocytes. 

Without  going  into  the  various  granule  stainings  so  thoroughly  brought  out  by 
Ehrlich,  we  shall  immediately  take  up  the  question  of  a  practical  classification  for  use 
in  making  a  differential  count.  As  the  Romanowsky  method  of  staining  (Wright, 
Leishman,  or  Giemsa)  gives  us  information  not  yielded  by  either  haematoxylin  and 
eosin  or  the  triacid,  the  points  of  differentiation  to  be  referred  to  in  that  which 
follows  is  with  blood  so  stained. 

In  considering  the  staining  affinities  of  different  parts  of  the  leuko- 
cytes, it  is  convenient  to  divide  such  into  basic  ones,  acid  ones,  and 
those  which  may  be  said  to  be  on  the  border  line  between  these — the 
so-called  neutrophilic  affinities. 

With  Wright's  stain  we  have  the  eosinophile  or  oxyphile  affinity  of  the 
granules  of  eosinophiles  for  acid  dyes,  in  this  case  eosin.  The  nuclei  and  baso- 


NORMAL  LEUCOCYTES  225 

phile  granules  have  affinities  in  greater  or  less  degree  for  basic  stains  (the  blue  and 
the  violet  shading  resulting  from  methylene  blue  as  modified  by  polychroming). 
With  the  granules  in  the  cytoplasm  of  the  polymorphonuclears  and  neutrophilic 
myelocytes,  and  to  a  less  extent  in  the  transitional,  we  have  a  staining  which  merges 
into  a  yellowish  red  on  the  one  extreme  and  into  a  lilac  on  the  other.  As  a  standard, 
neutrophilic  granules  should  be  a  mean  of  these  extremes. 

Not  only  by  reason  of  the  authority  of  Ehrlich,  but  because  such  a 
division  gives  all  variations,  which  can  then  be  combined  by  one  pre- 
ferring a  simpler  classification,  it  would  seem  proper  to  divide  the 
normal  leukocytes  into  hyaline  and  granular  cells.  Of  the  former  we 
have  the  lymphocytes,  the  large  mononuclears  and  the  transitionals.  Of 
the  latter  the  polymorphonuclears,  the  eosinophiles  and  the  mast  cells. 

HYALINE  LEUKOCYTES 

1.  Lymphocytes. — As  a  rule,  the  cells  of  this  type  are  about  the 
diameter  of  a  red  cell.     The  nucleus  is  generally  quite  round  but  may 
show  one  or  more  bulging  processes.     The  large  lymphocytes  are  rare 
in  the  blood  of  adults  but  make  up  about  10%  of  the  leukocytes  of  young 
children.     In  making  a  count  it  is  best  to  group  large  and  small  lympho- 
cytes under  one  heading  but  for  distinction  we  may  divide  them  into: 

(a)  Small  Lymphocytes. — These  are  small  round  cells  about  the  size 
of  a  red  corpuscle  with  a  large  centrally  placed,  deeply  violet  staining 
nucleus  and  a  narrow  zone  of  cytoplasm.     This  cytoplasm  may  not  be 
more  than  a  mere  crescentic  fringe.    This  is  the  type  of  lymphocyte 
which  makes  up  the  greater  proportion  of  the  leukocytes  in  chronic 
lymphatic  leukaemia.     At  times  these  cells  seem  to  be  composed  of 
nucleus  alone. 

(b)  Large  Lymphocytes. — These  are  of  the  same  type  as  small  lympho- 
cytes, but  possessing  more  cytoplasm.    The  nucleus,  while  round  and 
taking  a  fairly  deep  rich  violet  stain,  does  not  stain  so  deeply  as  the 
nucleus  of  the  small  lymphocytes.    The  cytoplasm  is  a  clear,  trans- 
lucent, pure  blue.     It  may  contain  pinkish  granules  known  as  azur 
granules,  but  these  are  of  rather  large  size  and  do  not  mar  the  glass- 
like  appearance.     They  are  from  9  to  i$n  in  diameter  and  are  com- 
mon in  children.     In  the  acute  lymphatic  leukaemias  they  at  times 
predominate. 

2.  Large  Mononuclears.— These  are  large  round  or  oval  cells  with 
a  nucleus  which  has  lost  the  richness  of  violet  staining  of  the  lymphocyte 
nucleus.     The  nucleus  is  furthermore  frequently  irregular  in  outline 
or  may  show  the  commencing  indentation  of  the  transitional  nucleus. 

is 


226  NORMAL   AND   PATHOLOGICAL  BLOOD 

There  is  not  that  sharp  distinction  between  nucleus  and  cytoplasm  that  exists  in 
the  lymphocytes.  The  cytoplasm  of  the  large  mononuclear  gives  the  impression  of 
opacity,  as  if  it  were  frosted  glass  instead  of  clear  glass.  The  neutrophile  mottling 
which  begins  to  appear  causes  a  disappearance  of  the  pure  blue  character  of  the  cyto- 
plasm of  the  lymphocyte.  It  is  principally  by  the  washed-out  staining  of  the  nucleus 
and  the  opaque  lilac  of  the  cytoplasm  that  we  differentiate  them  from  the  lympho- 
cytes. They  greatly  resemble  Turck's  irritation  forms  or  plasma  cells  and  may  be 
confused  with  myelocytes. 

3.  Transitionals. — These  appear  as  but  a  later  stage  in  the  decay 
of  the  large  mononuclears;  the  nucleus  is  more  indented,  frequently 
horseshoe-shaped,  and  has  a  washed-out  violet  shade  of  less  intensity 
than  that  of  the  large  mononuclears.  These  are  the  cells  so  often  dis- 
rupted in  smears.  The  old  view  that  the  transitional  was  the  pre- 
cursor of  the  polymorphonuclear  has  few  advocates  at  the  present 
time. 

While  it  may  be  convenient  to  consider  hyaline  cells  as  representing  different  stages 
in  development,  yet  from  a  standpoint  of  immunity  this  is  untenable.  The  large 
mononuclears  and  transitionals  are  the  cells  in  which  we  find  certain  animal  cells 
and  pigment  phagocytized,  as  is  the  case  in  malaria.  These  cells  are  the  macro - 
phages  of  Metchnikoff  and  are  probably  derived  from  the  bone  marrow. 

In  the  tropics  one  of  the  most  important  points  in  a  differential 
count  is  the  matter  of  an  increase  in  the  large  mononuclears  and 
transitionals,  both  of  which  seem  to  respond  to  the  same  stimulus, 
which  is  most  commonly  malaria  but  may  also  be  from  other  protozoal 
infections. 

From  a  practical  standpoint  I  always  group  them  together  and  as  a  matter  of  fact 
it  is  difficult  to  separate  a  large  mononuclear  showing  considerable  irregularity  of 
nucleus  from  a  transitional  with  less  marked  nuclear  indentation. 

The  lymphocytes  take  origin  from  the  lymphoid  tissue,  and  very 
probably  the  large  lymphocyte  is  a  younger,  more  immature  cell  than 
the  small  lymphocyte. 

Ehrlich  and  Naegeli  regard  the  large  mononuclears  as  of  myeloid  origin  while 
Pappenheim  considers  them  to  belong  to  the  group  of  lymphocytes. 

A  normal  percentage  of  large  mononuclears  and  transitionals  com- 
bined should  not  exceed  about  4%. 

GRANULE  CONTAINING  LEUKOCYTES 

In  addition  to  the  series  of  leukocytes  just  considered  we  have  present 
normally  in  the  blood  three  types  of  granular  cells  distinguished 
according  to  the  staining  affinity  of  their  granules.  These  are: 


ARNETH  INDEX  227 

1.  Polymorphonuclear  Leukocytes.— This  cell  normally  constitutes 
the  greater  proportion  of  the  leukocytes.     It  is  an  amoeboid,  actively 
phagocytic  cell,  about  10  or  i2/*  in  diameter,  and  is  the  microphage  of 
Metchnikoff. 

Bacteria  are  actively  phagocytized  by  this  cell,  and  it  is  the  cell  concerned  in 
determining  the  opsonic  power  of  blood  to  various  bacteria.  It  has  fine  lilac  gran- 
ules which  are  termed  neutrophilic  (epsilon  granules).  The  single  nucleus  is  rich  in 
chromatin  and  is  lobose  like  the  kernel  of  an  English  walnut;  frequently  it  resembles 
the  letter  z.  These  cells  are  derived  from  the  neutrophilic  myelocytes  of  the  bone 
marrow.  It  is  in  these  cells  that  the  glycogen,  or  iodophil  granules,  appear  in  certain 
suppurative  conditions. 

Arneth  Index.— A  great  deal  of  interest  has  been  aroused  in  the  so-called  Arneth 
index,  especially  in  connection  with  prognosis  in  tuberculosis  and  various  pyogenic 
infections.  The  basis  of  the  test  is  that  polymorphonuclears  showing  only  one  or 
two  nuclear  nodes  are  considered  immature  while  those  having  three,  four  or  five 
nuclear  nodes  possess  greater  phagocytic  power. 

A  normal  distribution  is  as  follows: 

Class  1  Class  II  Class  III  Class  IV  Class  V 

6%  35%  42%  16%  i% 

To  obtain  the  Arneth  index  add  to  the  sum  of  the  polymorphonuclear  percent- 
ages of  cells  containing  one  and  two  nodes  one-half  of  the  percentage  of  those  having 
three  nodes.  In  the  above  we  have  as  the  normal  Arneth  index  62. 

In  an  advanced  case  of  tuberculosis  we  might  have  an  index  of  79,  obtained  as 
follows : 

Class  I  Class  II  Class  III  Class  IV  Class  V 

20%  45%  28%  6%  i% 

2.  Eosinophile   Leukocytes. — These   are   very   striking   cells   with 
coarse  granules  staining  brilliantly  pink,  the  eosinophile,  oxyphile,  or 
acidophile  granules  (alpha  granules  of  Ehrlich).     The  cells  are  a  little 
larger  than  the  polymorphonuclears. 

The  normal  eosinophile  is  to  be  distinguished  from  the  eosinophilic  myelocyte  by 
possessing  two  distinct  lobes  in  the  nucleus.  At  times  we  find  three  nuclei.  The 
nucleus  of  the  myelocyte  is  round.  The  eosinophile  is  the  cell  so  frequently  increased 
in  infections  by  intestinal  animal  parasites. 

3.  Mast  Cells. — These  also  have  coarse  granules,  but  they  stain  a 
deep  violet  blue.     Hence  they  are  basophile  granules  (gamma  granules) . 
In  fresh  blood  these  granules  do  not  show  up  very  well,  thus  they  can  be 
distinguished  from  the  highly  refractile  granules  of  the  eosinophile. 


228  NORMAL   AND    PATHOLOGICAL  BLOOD 

The  trilobed  nucleus  stains  less  intensely  than  the  granules.     As  a  rule, 
the  mast  cell  is  about  the  size  of  a  polymorphonuclear. 
In  a  differential  count  of  normal  blood  we  find  about  the  following  percentages. 

Polymorphonuclears,  65  to  70%,  about  5000  per  cu.  mm. 
Small  lymphocytes,  20  to  30%  about  1500  per  cu.  mm. 
Large  lymphocytes,  2  to  6%,  about  200  per  cu.  mm. 
Large  mononuclears,  i  to  2%,  about  100  per  cu.  mm. 
Transitionals,  2  to  4%,  about  200  per  cu.  mm. 

Eosinophiles,  i  to    2%,  about    100  per  cu.  mm. 

Mast  cells,  M  to  >£%,  about      25  per  cu.  mm. 

NOTE:  The  lymphocyte  percentage  of  infants  is  about  60. 

DIFFERENTIAL  COUNT 
« 
In  making  a  differential  count  I  would  recommend  the  following 

from  the  directions  of  Schilling-Torgau. 

It  will  be  remembered  that  considerable  interest  was  raised  a  few  years  ago  in  what 
was  termed  the  Arneth  index.  In  this  the  more  normal,  more  mature,  better  resist- 
ing polymorphonuclears  were  considered  to  have  three  or  four  lobes  to  the  nuclear 
structure,  even  occasionally  five.  The  immature  cells  had  only  one  or  at  most  two 
lobes  to  the  nucleus.  The  index  was  obtained  by  adding  the  percentages  of  cells 
showing  one  and  two  lobes  to  one-half  the  percentage  of  those  with  three  lobes.  As 
will  be  understood  a  high  percentage  of  these  immature  cells  was  unfavorable  in  prog- 
nosis. These  cells  are  graded  from  left  to  right  I,  II,  III,  IV,  V,  as  to  separate 
masses  in  the  nucleus,  so  that  when  the  percentage  is  shoved  or  displaced  to  the  left 
it  indicates  an  increase  in  the  immature  cells. 

Schilling-Torgau  divides  his  polymorphonuclears  into  i.  the  myelocyte  which  is 
always  of  course  a  pathological  cell;  2.  the  immature  form  polymorphonuclear.  In 
this  there  is  a  close  resemblance  to  the  neutrophile  myelocyte  but  there  is  a  nuclear 
indentation  instead  of  the  round  nucleus  of  the  myelocyte.  It  is  this  cell  which  often 
puzzles  us  as  to  whether  to  regard  it  as  a  true  myelocyte.  It  is  the  metamyelocyte 
of  many  authorities.  3.  Between  the  mature  or  segmented  polymorphonuclear 
and  the  immature  one  or  metamyelocyte  we  have  what  may  be  designated  the  band 
form  nucleated  one.  These  show  the  type  of  nucleus  which  one  is  familiar  with  in 
the  nucleus  of  the  transitional.  4.  The  mature,  multilobed  or  segmented  nucleus  of 
the  typical  polymorphonuclear. 

It  would  seem  that  if  all  laboratory  workers  would  agree  upon  some 
single  method  of  recording  differential  counts  it  would  be  advantageous. 

In  the  differential  count  he  not  only  divides  up  the  polymorphonuclears  but 
makes  no  separation  of  small  from  large  lymphocytes.  Although  I  have  always 
divided  lymphocytes  into  large  and  small  ones  I  believe  it  unnecessary  and  imprac- 
tical and  shall  henceforth  group  all  such  cells  in  one  grouping.  The  statement  that 
large  mononuclears  and  transitionals  are  cells  of  a  similar  origin,  type  and  significance 
has  always  been  my  idea. 


PATHOLOGICAL  LEUCOCYTES 
SCHEME  OF  SCHILLING-TORGAU 


229 


Type  of  cell  Normal                      Percentage  , 

percentage 

1.  Mast  cells.  x                         x  0 

2.  Eosinophiles.  ,                           i   e 

f  a.  myelocytes.  o                         0.5 

Neutro-      '  b'  immature    fonns  (metamye- 

'  ~ 


philes                     ocyes.  o  5.0 

I  c.  bandform  (Stabkernige).  4  I3.5 

[  d.  multilobed  (Segmentkernige)  .  63  64.0 

4.  Lymphocytes.  23  10.5 

5.  Large  mononuclears  and  transitionals.  6  4.0 


PATHOLOGICAL  LEUKOCYTES 

The  leukocytes  which  are  found  in  the  peripheral  circulation  only 
in  pathological  conditions  are: 

1.  Neutrophilic  Myelocytes. — The   common   type  is   a  large   cell 
with  a  large  centrally  placed,  feebly  staining  nucleus. 

This  may  be  recognized  by  the  difficulty  of  distinguishing  the  nucleus  from 
the  cytoplasm,  there  being  no  sharp  line  separating  these  parts  of  the  cell.  They 
imperceptibly  merge  into  one  another.  They  differ  from  a  large  mononuclear  in 
that  the  cytoplasm  is  distinctly  dotted  with  neutrophile  granules  and  that  we 
cannot  make  out  a  distinct  line  of  separation  of  a  slightly  irregular  or  indented 
nucleus  from  the  surrounding  slightly  neutrophilic  cytoplasm.  Cornil  has  described 
a  very  large  myelocyte  with  eccentrically  placed  nucleus  and  neutrophilic  granules. 

Myelocytes  are  at  times  found  with  both  basophilic  and  neutrophilic 
granules,  and  may  rarely  be  seen  to  have  all  three  kinds  of  granules  on  a 
single  myelocyte,  acidophile,  basophile,  and  neutrophile. 

2.  Eosinophilic    Myelocyles. — These    can    be    distinguished    from 
normal  eosinophiles  by  their  possessing  a  single  round  nucleus,  not 
bilobed.     These  myelocytes  may  be  as  large  as  a  normal  eosinophile, 
but  frequently  are  no  larger  than  a  red  cell. 

The  neutrophile  myelocyte  is  characteristic  of  spleno-myelogenous  leukaemia,  the 
eosinophile  one  of  myelogenic  leukaemia.  The  occurrence  of  an  occasional  myelocyte 
is  frequently  noted  in  conditions  having  a  leukocytosis.  In  diphtheria  their  presence 
in  numbers  is  of  bad  prognostic  import.  Myelocytes  are  of  diagnostic  importance 
in  metastases  of  malignant  tumors. 

3.  The  Irritation  Cell  of  Tiirck,  or  Plasma  Cell. — This  cell  has  a 
faintly  staining,  eccentrically  placed  nucleus,  and  a  dark  opaque  blue, 
frequently  vacuolated,  cytoplasm.     They  are  usually  recorded  as  large 


230  NORMAL  AND   PATHOLOGICAL  BLOOD 

mononuclears.     Tiirck  supposed  them  to  appear  in  the  circulation  as 
the  result  of  bone-marrow  irritation. 

4.  Myeloblasts. — These  cells  are  found  in  myeloid  leukaemia  and 
though  often  mistaken  for  lymphocytes  or  large  mononuclears  they  are 
of  marrow  origin.     They  are  the  lymphoid  cells  of  the  marrow  and  are 
the  parent  cells  of  myelocytes.     The  nucleus  stains  more  intensely 
than  that  of  the  large  mononuclear  and  the  cytoplasm  is  more  deeply 
blue  stained  than  that  of  the  large  lymphocyte.     They  also  contain 
three  or  four  nucleoU. 

Pyronin  methyl-green  staining  is  best  for  demonstrating  the  nuclei. 

5.  Pathological  Large  Lymphocytes. — These  are,  as  a  rule,  much 
larger  than  normal  large  lymphocytes  and  show  poorer  staining  of  both 
nucleus  and  cytoplasm.     The  nuclei  often  show  the  appearance  of 
division  into  two  or  more  lobes,  thus  showing  the  characteristics  of 
Rieder  cells. 

They  may  be  confused  with  large  mononuclears  but  are  considered  to  be  derived 
from  the  germinal  centers  of  various  lymphoid  tissues.  They  are  found  in  leukaemic 
and  pseudo-leuksemic  conditions. 

6.  Megakaryocytes. — These  are  the  giant  cells  of  the  bone  marrow 
and  are  but  rarely  found  in  the  blood.     The  nucleus  is  gnarled. 

BLOOD  PLATELETS 

These  are  normally  present  in  blood  in  the  number  of  about  350,000  per  cubic  milli- 
meter. They  disintegrate  very  quickly  after  the  blood  is  withdrawn.  Wright  has 
demonstrated  that  they  are  pinched-off  projections  of  giant  cells  of  the  bone  marrow. 
They  consist  only  of  protoplasm,  no  nuclear  material.  They  do  not  contain  haemo- 
globin. In  conditions  where  giant  cells  are  less  abundant,  as  in  pernicious  anaemia, 
the  blood  platelets  are  less  abundant.  In  myelogenous  leukaemia  they  are  very  abun- 
dant. They  vary  in  size  from  2  to  5/1  according  as  a  larger  or  smaller  pseudopod  of  a 
giant  cell  has  been  broken  off.  Stained  with  Wright's  stain,  they  are  more  purplish 
than  blue  and  show  thread-like  projections.  They  are  often  mistaken  for  the  proto- 
zoal  causes  of  various  diseases.  Especially  are  they  confused  with  malarial  para- 
sites when  lying  on  a  red  cell.  The  blood  plate  has  no  brick-red  chromatic  material; 
it  is  purplish  rather  than  blue,  and  has  no  pigment  grains.  It  is  advisable  to  com- 
pare these  isolated  blood  plates  with  the  larger  or  smaller  aggregations  scattered 
about  the  smears.  In  this  way  their  true  character  is  apparent.  In  addition  to 
blood  platelets,  which  in  fresh  blood  can  only  be  observed  when  a  fixative  is  used,  we 
have  other  confusing  bodies. 

The  hamokonia  of  Muller  are  small,  highly  refractile  bodies  showing  active 
oscillatory  movement.  They  are  supposed  to  be  cast-off  granules  of  eosinophiles  or 


EOSINOPHILIA  231 

other  leukocytes,  or  possibly  derived  from  nuclei.  As  this  blood  dust  or  haemokonia 
is  found  in  a  marked  degree  in  lipaemia  it  may  be  that  the  particles  are  fat.  It  is 
interesting  that  this  lipaemia  is  absent  after  the  taking  of  large  quantities  of  fat  in 
cases  with  serious  pancreatic  trouble.  The  serum  of  a  normal  individual  is  rather 
turbid  after  slight  indulgence  in  butter.  Pinched-off  fragments  of  red  cells  may 
also  appear  as  possible  protozoal  bodies. 

LEUKOPENIA 

This  is  a  term  used  to  designate  a  reduction  in  the  normal  number 
of  leukocytes.  A  leukocyte  count  of  5000  would  represent  a  slight 
leukopenia;  one  of  2000,  a  marked  leukopenia.  In  the  latter  stages 
of  typhoid,  and  in  acute  miliary  tuberculosis,  we  expect  a  moderate 
leukopenia.  Glandular  tuberculosis  may  give  a  very  marked  leuko- 
penia. Tuberculous  peritonitis  will  show  moderate  leukopenia  or  a 
normal  count. 

The  leukopenia  of  typhoid  is  moderate  and  is  often  preceded  in  the  first  few  days 
by  a  moderate  neutrophile  leukocytosis.  Later  on  we  have  a  decided  increase  in  the 
lymphocytes.  A  marked  diminution  or  absence  of  eosinophiles  is  so  characteristic 
that  any  increase  in  eosinophilic  percentage  negatives  a  diagnosis  of  typhoid. 

Paratyphoid  gives  a  similar  blood  picture. 

Chronic  alcoholism  and  chronic  arsenic  poisoning  cause  a  reduction  in  the  number 
of  the  white  cells.  Pernicious  anaemia  especially  the  aplastic  type  shows  a  marked 
leukopenia,  as  is  also  the  case  with  Banti's  disease.  Two  tropical  diseases,  kala-azar 
and  dengue,  show  a  marked  leukopenia,  the  counts  often  being  below  2500.  During 
the  apyrexial  period  of  malaria  we  may  have  a  white  count  of  5000. 

It  has  recently  been  claimed  that  a  leukopenia  with  a  coincident 
marked  reduction  in  the  lymphocytes  is  characteristic  of  measles  and 
that  this  occurs  several  days  before  the  Koplik  spots  appear. 

Kocher  notes  that  in  exophthalmic  goiter  the  leukocyte  count  is  considerably 
diminished  and  that  the  polymorphonuclears  are  not  much  more  than  one-half  the 
usual  percentage  while  the  percentage  of  the  lymphocytes  is  almost  double  the 
normal. 

X-ray  treatment  tends  to  destroy  leukocytes  in  the  exposed  region,  especially 
polymorphonuclears.  The  small  lymphocytes  are  least  affected. 

EOSINOPHILIA 

Where  the  eosinophiles  are  increased  to  5%,  we  have  a  moderate 
eosinophilia.  In  some  cases  of  infection  with  intestinal  parasites, 
especially  hook-worms,  but  also  from  other  parasites,  as  round  and 
whip-worms,  we  may  have  an  eosinophilia  of  30  to  50%.  In  Guam, 
among  the  natives,  it  is  difficult  to  find  an  eosinophile  count  under  15.% 


232  NORMAL  AND   PATHOLOGICAL  BLOOD 

The  eosinophilia  tends  to  disappear  when  the  anaemia  becomes  very 
severe. 

Echinococcus  infection  has  an  eosinophilia  which  disappears  when  the  cyst  is 
removed.  Continuance  of  the  eosinophilia  indicates  that  all  cysts  were  not  gotten 
rid  of. 

The  eosinophilia  of  trichinosis  is  best  known,  and  a  combination  of 
this  blood  finding  with  fever  and  marked  pains  of  muscles,  would 
justify  the  excision  of  a  piece  of  muscle  for  examination  for  encysted 
embryos. 

In  true  asthma  eosinophilia  is  marked,  and  its  absence  is  of  value  in  indicating 
other  causes  for  the  condition.  Certain  skin  diseases,  especially  pemphigus,  show 
eosinophilia.  Blastomycoses  are  usually  found  to  show  eosinophile  increase. 

Eczema  and  psoriasis  are  not  apt  to  give  more  than  3  or  4%  eosino- 
philes.  A  rather  high  degree  of  eosinophilia  is  found  in  mycosis 
fungoides. 

Scabies  also  gives  an  eosinophilia. 

The  proportion  of  eosinophiles  in  the  blood  of  children  is  greater 
than  in  that  of  adults. 

Increase  of  both  eosinophiles  and  mast  cells  is  found  in  myelogenous  leukaemia. 

An  eosinophilia  tends  to  appear  following  splenectomy.  With  a  Wright  stain 
showing  acid  tendencies  one  may  count  polymorphonuclears  as  eosinophiles  unless 
noting  smaller  size  of  granules. 

LEUKOCYTOSIS 

It  is  to  an  increase  in  the  polymorphonuclears  that  this  term  is 
usually  applied,  the  term  lymphocytosis  or  eosinophilia  being  employed 
where  white  cells  of  eosinophile  or  lymphocyte  nature  are  increased. 
We  have  physiological  leukocytosis  in  the  latter  weeks  of  pregnancy, 
also  in  the  new-born,  and  in  connection  with  digestion. 

Pathological  Leukocytosis.— Pneumonia.  In  this  disease  we  have  a 
leukocytosis  of  20,000  to  30,000  or  higher.  The  eosinophiles  are  al- 
most absent.  A  normal  leukocyte  count  in  pneumonia  makes  a  prog- 
nosis unfavorable. 

The  leukocyte  count  drops  about  the  time  of  the  crisis,  and  with  the  reappearance 
of  eosinophiles  is  a  favorable  sign. 

Toxaemic  conditions  as  uraemia,  diabetic  coma  and  poisoning  by  CO2  tend  to  show 
a  leukocytosis. 


LEUCOCYTOSIS 


233 


Septic  processes.  The  leukocyte  count  is  of  great  value,  especially 
when  we  obtain  a  leukocytosis  with  80  to  90%  of  polymorphonuclears, 
as  in  appendicitis,  cholecystitis,  or  other  suppurative  conditions. 
Read  article  under  the  blood  cultures,  Chap.  XXIX.  A  marked 
leukocytosis  is  of  diagnostic  importance  in  acute  ulcerative  endocarditis 
provided  it  is  not  fulminant  in  type. 


FIG.  55.— Leukocytosis  (40,000);  sixteen  polymorphonuclears  in  field.     (Cabot.) 

According  to  Cabot,  leukocytosis  varies  in  infections  as  follows: 

1.  Severe  infection— good  resistance;  early,  marked  and  persistent  leukocytosis. 

2.  Slight  infection — slight  resistance;  leukocytosis  present,  but  not  marked. 

3.  In  fulminating  infections  we  may  have  no  increase  in  whites,  but  a  higher  per- 
centage of  polymorphonuclears. 

4.  Slight  infection  and  good  resistance  may  not  be  productive  of  leukocytosis. 

It  is  in  connection  with  the  question  of  operation  in  appendicitis  or  similar  condi- 
tions that  the  matter  of  a  leukocyte  count  is  of  prime  importance.  If  there  be  a 
leukocytosis  but  with  less  than  75%  of  polymorphonuclears  it  indicates  an  infection 
of  little  virulence  or  a  walled-off  process  with  an  exacerbation.  It  is  difficult  to  form 
an  opinion  when  the  polymorphonuclears  are  under  80%.  Leukocytosis  with  poly- 
morphonuclear  percentage  of  85  to  90  indicates  immediate  operation;  percentages 
over  90  point  to  peritonitis  and  if  with  such  percentages  of  polymorphonuclears  there 
is  absence  of  leukocytosis  the  prognosis  is  grave. 

The  blood  of  cases  with  malignant  tumors  tends  to  show  a  moderate 
leukocytosis  except  in  epithelioma  of  the  skin.  When  a  cancer  is 
ulcerating  quite  a  high  white  count  may  be  obtained. 

Spirochaete  fevers,  as  relapsing  fever,  may  give  a  leukocytosis  of  from  25,000  to 
50,000. 

Smallpox,  especially  at  time  of  pustulation,  plague,  scarlet  fever, 
and  liver  abscess  give  a  leukocytosis  of  from  12,000  to  15,000. 


234  NORMAL   AND   PATHOLOGICAL  BLOOD 

Smallpox  often  shows  a  very  large  percentage  of  very  characteristic  large 
nuclears. 

The  leukopenia  and  lymphocyte  increase  in  measles  are  important  points  in 
differentiating  it  from  scarlatina. 

Influenza  shows  a  leukopenia  at  first,  then  a  leukocytosis  and,  follow- 
ing the  fall  in  fever,  a  second  lowering. 

With  meningitis  counts  of  25,000  are  not  unusual,  in  abscess  of  the  brain  the  white 
count  rarely  exceeds  i5,'ooo. 

Poliomyelitis  and  polioencephalitis  give  a  slight  leukocytosis  during 
the  febrile  accession. 

Erysipelas  and  epidemic  cerebrospinal  meningitis  also  give  a  leukocytosis  of  from 
1 5,000  to  20,000.  In  malignant  diseases  we  sometimes  have  a  moderate  leukocytosis. 
Rogers  states  that  in  liver  abscess,  with  a  leukocytosis  of  15,000  to  20,000,  we  have 
only  about  75  to  77%  of  polymorphonuclears — there  being  also  a  moderate  increase 
in  the  percentage  of  large  mononuclears. 

Drugs  such  as  antipyrin  may  give  a  leukocytosis.  The  leukocyte  increase  of 
pilocarpine  is  rather  a  lymphocytosis.  Cinnamate  of  soda,  sodium  nucleate  bacterin 
injections  and  turpentine  have  been  used  in  kala-azar  to  increase  leukocytes. 

LYMPHOCYTOSIS 

Of  course,  the  disease  in  which  we  have  the  most  marked  lymphocy- 
tosis is  lymphatic  leukaemia. 

The  lymphocytosis  of  typhoid  fever  has  been  taken  up  under  leukopenia. 
Whooping-cough  may  give  a  lymphocytosis  of  20,000  to  30,000. 

Young  children  have  normally  an  excessive  proportion  of  lymphocytes  even  to  a 
reversal  of  the  polymorphonuclear-lymphocyte  relation  of  adults.  This  is  apt  to  be 
particularly  marked  in  hereditary  syphilis.  Enlarged  tonsils  may  give  rise  to  a 
lymphocytosis  of  10,000  to  15,000  when  more  than  50%  of  the  white  cells  will  be 
lymphocytes.  Rickets  and  scurvy  give  a  lymphocytosis. 

In  pellagra  there  is  a  moderate  lymphocytosis,  averaging  34%  in  about  a  normal 
count. 

Varicella  and  mumps  may  also  give  an  increase  in  the  percentage  of 
lymphocytes. 

Malta  fever  is  a  disease  which  may  show  quite  a  lymphocyte  increase,  this  going 
with  a  reduction  in  polymorphonuclears. 

INCREASED  LARGE  MONONUCLEARS 

In  tropical  work  we  combine  the  large  mononuclears  and  transitionals 
in  a  differential  count.  They  are  the  phagocytes  of  animal  cells  or 


PRIMARY  ANEMIAS  235 

parasites.  The  disease  in  which  their  increase  is  best  recognized 
is  malaria  and  an  increase  to  15%  where  the  blood  shows  moderate 
leukopenia  is  very  significant.  The  melaniferous  leukocytes  of  malaria 
are  cells  of  this  type. 

Other  protozoal  infections,  as  kala-azar,  trypanosomiasis  and  amcebiasis  cause  it. 
Filterable  virus  diseases  may  show  a  mononuclear  increase,  thus  yellow  fever  and 
dengue  both  give  an  increase  about  the  fifth  or  sixth  day. 

In  Banti's  disease  there  is  an  increase  in  cells  of  this  type  and  a  transitional  in- 
crease is  reported  for  Hodgkin's  disease. 

DISEASES  IN  WHICH  THERE  is  A  NORMAL  LEUKOCYTE  COUNT 

Uncomplicated  tuberculosis,  influenza,  Malta  fever,  measles,  try- 
panosomiasis, malaria,  syphilis,  and  chlorosis. 

In  malaria  we  have  a  leukocytosis  at  the  time  of  the  rigor,  while  during  the  apyrexial 
period  there  is  a  moderate  leukopenia.  In  malaria  we  have  a  marked  increase  in  the 
percentage  of  the  large  mononuclears  and  transitionals.  These  may  form  from  25% 
to  35%  of  the  leukocytes.  When  bearing  particles  of  pigment  they  are  known  as 
melaniferous  leukocytes — macrophages  which  have  ingested  malarial  material.  In 
dengue,  at  the  time  of  the  terminal  rash,  we  may  have  as  great  a  percentage  of  large 
mononuclears.  In  this  disease,  however,  we  have  a  great  diminution  of  polymor- 
phonuclears  from  the  start  (25  to  40%).  Instead  of  a  large  mononuclear  we  have 
at  the  onset  a  lymphocytic  increase.  There  is  an  increase  of  large  mononuclears  in 
trypanosomiasis. 

The  white  count  is  about  normal  in  uncinariasis  (Ashford's  average 
was  7800).  Some  have  reported  a. leukopenia  in  severe  cases. 

While  eosinophilia  is  the  most  marked  feature  in  hook-worm  disease  yet  in  very 

severe  cases  it  may  be  absent. 

•- 

THE  PRIMARY  ANAEMIAS 

Chlorosis.— In  chlorosis  it  is  the  reduction  of  haemoglobin  with  the 
slight  numerical  variation  from  normal  of  the  red  cells  that  makes  for 
a  diagnosis.  The  color  index  is  very  low.  There  is  nothing  abnormal 
about  the  leukocytes.  Microcytes  may  be  present,  and  very  occasion- 
ally a  normoblast.  Macrocytes  and  megaloblasts  are  always  absent. 
Blood  of  chlorotics  is  very  pale  and  very  fluid  and  coagulates  rapidly, 
hence  frequency  of  thrombosis. 

Spleen,  liver,  and  lymph  glands  as  a  rule  normal. 

Simple  Primary  Anaemia.— This  condition  is  not  recognized  by  many  authors, 
but  is  a  convenient  term  under  which  to  group  anaemias  which  are  neither  chlorosis 


236  NORMAL  AND   PATHOLOGICAL  BLOOD 


nor  pernicious  anaemia  and  for  which  no  assignable  cause  can  be  designated.  It 
is  a  secondary  anaemia  without  a  cause.  In  it  color  index  is  about  normal,  there  is 
no  change  in  the  leukocytes  and  cases  go  on  to  recovery. 

Pernicious  Anaemia. — In  pernicious  anaemia  we  obtain  a  very 
fluid,  but  normally  colored  drop  of  blood  upon  puncture.  The  yellow 
marrow  of  the  long  bones  is  transformed  into  a  soft,  bright  red  lymphoid 
tissue,  smears  from  which  show  great  numbers  of  megaloblasts. 

Areas  of  fatty  degeneration  are  characteristic,  especially  the  tiger-lily  spots  in 
the  heart  muscle.  Iron-containing  pigment  (hemosiderin)  is  found  in  the  liver, 
spleen,  and  kidneys.  Areas  of  degeneration  in  the  spinal  cord  may  account  for  nerv- 


FIG.  56. — Pernicious  anaemia.     M.m,   Megaloblasts;   n,   normoblast;   s,   stippling 
(punctate  basophilia).     (Cabot.) 

ous  symptoms.  The  red  cells  frequently  fall  below  2,000,000  with  patients  going 
about.  Cases  have  been  reported  with  counts  under  200,000.  The  color  index  is 
high.  Megaloblasts  are  the  most  characteristic  qualitative  change  in  the  red  cells. 

Megaloblastic  crises  may  at  certain  times  show  enormous  numbers 
of  megaloblasts.  Cases  often  present  remissions  in  which  no  megalo- 
blasts can  be  found.  In  such  cases  the  presence  of  many  macrocytes 
should  prevent  an  examiner's  reporting  against  a  pernicious  anaemia 
previously  diagnosed. 

Poikilocytosis,  polychromatophilia,  and  stippling  are  also  features  of  the  disease. 
Normoblasts  are  far  less  frequent  than  megaloblasts  and  there  is  usually  a  moderate 
lymphocytosis.  Myelocytes  may  be  present,  but  their  precursors,  the  myeloblasts, 
are  probably  more  frequently  met  with. 

Cases  of  pernicious  anaemia  show  remissions  during  which  the  patient  is  appar- 
ently on  the  road  to  recovery.  Such  improvements  are  only  temporary.  The 
remissions  may  last  from  two  months  to  possibly  three  or  four  years.  Especially 


SECONDARY  ANAEMIAS  237 

in  the  anaemia  of  Dibothriocephalus  latus  do  we  have  a  picture  of  pernicious  anaemia. 
It  is  supposed  to  be  due  to  a  toxin  present  in  the  heads  of  these  tape-worms. 

In  pernicious  anaemia  there  is  usually  an  absence  of  free  HC1,  which 
causes  it  often  to  be  confused  with  gastric  cancer.  Cases  of  chronic 
nephritis  are  also  often  much  like  pernicious  anaemia  but  most  difficult 
of  differentiation  are  affections  of  the  spinal  cord,  such  as  tabes,  etc., 
the  neurological  manifestations  of  pernicious  anaemia  causing  the 
confusion. 

Blood  changes  more  or  less  like  those  of  pernicious  anaemia  have  at  times  been 
noted  in  children  with  tuberculosis  of  bovine  nature.  The  human  strain  of  T.B. 
does  not  seem  to  produce  such  changes. 

An  acute  disease  showing  a  rapidly  developing  anaemia  of  the  pernicious  anaemia 
type  is  Oroya  fever  in  which  the  bone  marrow  seems  especially  involved. 

SECONDARY  ANEMIAS 

These  are  the  anaemias  which  can  be  definitely  traced  to  some  dis- 
ease not  of  the  haemopoietic  system. 

There  are  two  main  groups — those  following  haemorrhage  and  those 
secondary  to  various  diseases.  If  the  haemorrhage  is  sudden  and  great, 
the  resulting  condition  is  one  of  oligochromaemia — chlorotic  in  type. 
Normoblasts  are  usually  found  after  the  third  day. 

The  low  Hb.  percentage  is  apt  to  continue  for  several  weeks.  There  is  also  an 
increase  in  the  percentage  of  polymorphonuclears. 

It  is  a  question  whether  prolonged  operation  or  those  requiring  narcosis  are 
justified  where  the  reduction  in  Hb.  is  under  40%.  (According  to  Miculicz,  30% 
is  the  minimum). 

Where  the  loss  of  blood  is  gradual,  as  in  gastric  cancer  or  severe  haemorrhoids 
the  picture  may  more  nearly  approach  that  of  pernicious  anaemia.  Secondary 
anemias  usually  show  a  moderate  leukocytosis.  In  chronic  nephritis  and  prolonged 
suppurative  conditions  normoblasts  and  macrocytes  are  rare— moderate  poikilo- 
cytosis  with  the  presence  of  many  microcytes  being  the  rule. 

In  fatal  ansmia  from  chronic  acetanilide  poisoning  high  color  index,  macrocytes 
and  megaloblasts  have  been  noted. 

In  some  secondary  anaemias,  as  in  syphilis,  carcinoma,  and  tuber- 
culosis, we  have  a  chlorotic  color  index  (chloro-anaemias). 

In  secondary  anaemias  polychromatophilia,  poikilocytosis,  and  punc- 
tate basophilia  (stippling)  may  be  present.  This  latter  is  very  marked 
in  lead  poisoning,  but  in  certain  cases  of  malarial  cachexia  it  may  be 
equally  prominent.  The  only  form  of  nucleated  red  cell  seen  is  the 
normoblast,  in  very  small  numbers,  or  it  may  not  be  present. 


238  NORMAL  AND   PATHOLOGICAL  BLOOD 


Megaloblasts  are  practically  never  seen,  except  in  some  of  the  very  severe  para- 
sitic anaemias,  as  the  broad  Russian  tape-worm  infection.  The  red  cells  generally 
number  between  2,000,000  and  4,000,000,  thus  differentiating  chlorosis.  The 
leukocytes  are  frequently  increased  to  15,000.  In  the  anaemia  of  splenic  anaemia 
there  is  a  marked  leukopenia.  In  anaemias  from  malignant  tumors  the  color  index 
is  usually  of  the  chlorotic  type — the  haemoglobin  content  of  the  red  cells  being  more 
affected  than  the  number.  Normoblasts  are  usually  present,  and  this  finding  may 
differentiate  gastric  cancer  from  ulcer.  In  bone  marrow  metastases  megaloblasts 
may  be  expected.  Myelocytes  and  so-called  tumor  cells  (large  cells  with  faintly 
staining  vacuolated  nuclei  and  but  little  cytoplasm)  may  also  be  found.  As  a  rule, 
there  is  a  moderate  leukocytosis  in  malignant  disease.  Eosinophiles  may  be  largely 
increased  in  sarcoma. 

THE  LEUKAEMIAS 

It  is  in  the  leukaemias  that  we  have  the  greatest  increase  in  the  num- 
ber of  white  cells.  These  cases  show  more  or  less  anaemia,  but  we  may 
have  cases  of  myelogenous  leukaemia  showing  250,000  leukocytes  per 
cubic  millimeter  without  particular  change  in  the  red  cells.  The  more 
marked  the  red-cell  change  the  more  severe  the  condition. 

There  are  two  well-defined  types  of  leukaemia,  the  lymphatic  and  the  spleno- 
myelogenous.  It  must  be  borne  in  mind,  however,  that  while  a  greater  change  in 
the  lymphatic  glands  may  produce  the  lymphatic  type,  yet  even  in  such  cases  we 
expect  to  find  alteration  in  bone  marrow  and  spleen;  that  is,  there  is  a  general  involve- 
ment of  the  haemopoietic  system  in  all  leukaemias,  the  activity  being  most  marked  in 
spleen  and  bone  marrow  in  certain  cases  and  in  lymphatic  glands  in  others. 

Myelogenous  leukaemia  is  a  very  rare  disease,  about  five  times  as 
rare  as  pernicious  anaemia.  Lymphoid  leukaemia  is  still  more  rare. 

Splenomyelogenous  Leukaemia  (myeloid  leukaemia). — The  differen- 
tiation of  the  blood  picture  of  this  disease  from  leukocytosis  does  not 
depend  on  the  number  of  leukocytes,  but  on  the  presence  and  large 
proportion  of  myelocytes.  We  expect  both  neutrophilic  and  eosino- 
philic  myelocytes  in  myeloid  leukaemia — the  proportion  of  these  varies, 
but,  as  a  rule,  the  neutrophilic  one  is  the  common  one.  The  blood  in 
advanced  cases  is  milky  and  shows  a  most  marked  buffy  coat.  The 
marrow  is  largely  replaced  by  a  yellow  pyoid  material.  The  spleen 
may  weigh  10  pounds. 

The  leukocyte  count  is  on  the  average  from  200,000  to  500,000.  Cases  are  re- 
ported of  more  than  1,000,000  white  cells.  The  neutrophilic  myelocytes  make 
up  about  30  to  40%  of  these  and,  about  equal  in  number,  are  found  the  polymor- 
phonuclears,  while  the  percentage  of  the  lymphocytes  is  decreased  (2  to  5%)  and 
normal  eosinophiles,  eosinophilic  myelocytes,  and  large  mononuclears  make  up  the 
remaining  percentages.  We  usually  have  great  numbers  of  normoblasts.  Megalo- 


. 


LEUKAEMIA 


239 


blasts  may  rarely  be  found.    The  red  count  is  usually  about  2,500,000  and  the 
color  index  low. 

Lymphatic  Leukaemia.— In  this  we  have  glandular  enlargements, 
but  not  such  large  masses  as  in  Hodgkin's  disease.  •  The  red  cells  are 
usually  reduced  about  one-half  and  the  color  index  is  a  little  below 
normal.  Normoblasts  are  rarely  found.  Myelocytes,  as  a  rule,  are 
absent,  but  may  amount  to  5%  of  the  leukocytes.  The  predominating 
leukocyte  (75  to  98%)  is  the  small  lymphocyte.  In  acute  lymphatic 
leukaemia  the  large  lymphocytes  predominate. 


FIG.  57. — Myelogenous  leukaemia,     m,  Myelocyte;  p,  polymorphonuclear;  b,  mast 
cell;  n,  normoblast.     (Cabot.) 

These  however  are  pathological  and  differ  from  the  large  lymphocyte  in  not 
having  azur  granules  and  the  nucleus  stains  poorly  and  is  often  indented.  The 
leukocyte  count  is  never  so  great  as  in  myeloid  leukaemia,  rarely  exceeding  125,000. 

Pseudoleukaemia.— Hodgkin's  disease  is  usually  considered  as  a 
disease  with  marked  glandular  enlargements,  but  with  a  negative  blood 
picture,  or  at  any  rate  only  a  moderate  leukocytosis  with  a  relative 
increase  of  lymphocytes. 

The  glands  in  this  condition  do  not  soften  and  on  section  show  diffuse  hyper- 
plasia  of  cells  of  the  endothelial  type.  Eosinophiles  are  also  abundant  in  the  sec- 
tions. Connective-tissue  increase  and  small  necrotic  areas  are  also  features. 

The  red  cells  are  usually  above  3,000,000.  It  has  been  considered  that  an  in- 
creased percentage  of  transitionals  (10  to  15%),  should  a  leukopenia  coexist,  is 
characteristic. 

Undoubtedly  the  view  that  so-called  lymphosarcomata,  lymphatic 
leukaemia,  and  Hodgkin's  disease  merge  into  one  another  and  that  they 


240  NORMAL   AND   PATHOLOGICAL  BLOOD 

represent  a  malignant  cell  formation  in  the  haemopoietic  system  is 
conservative  one  to  take. 


A  certain  proportion  of  cases  of  Hodgkin's  disease,  however,  show  endothelial 
cell  proliferation  and  a  chronic  fibroid  change. 

In  Kundrat's  lymphosarcoma  we  have  a  neutrophile  leukocytosis 
and  a  diminution  of  the  lymphocytes.  The  spleen  and  liver  are  rarely 
involved. 

Another  condition  with  swelling  of  the  lymphatic  glands,  which  do  not  however 
fuse,  is  the  so-called  granulomatosis. 


FIG.  58. — Lymphatic  leukaemia.     />,  polymorphonuclear;  m,  megaloblast;  e,  eosino- 
phile.     Twenty-one  lymphocytes  in  this  field.     (Cabot.) 


In  this  we  have  a  polymorphonuclear  leukocytosis  of  from  20,000  to  50,000  with 
an  increase  in  the  percentage  of  eosinophiles.  The  lymphocytes  are  absolutely  and 
relatively  decreased.  In  granulomatosis  there  is  no  tendency  to  haemorrhage. 

Splenomegaly. — The  best  known  anaemia  associated  with  splenic 
enlargement  is  Band's  disease. 

Banti's  disease  also  has  a  very  low  color  index  and  leukopenia.  In 
this  the  primary  affection  is  of  the  spleen  which  becomes  greatly  en- 
larged. The  accompanying  cirrhosis  of  the  liver  with  its  symptoms  of 
ascites,  etc.,  differentiate  it.  Splenectomy  often  cures  the  disease. 
The  leukopenia  is  one  showing  not  only  a  diminution  of  polymorphonu- 
clear percentage  but  of  cells  of  the  lymphocyte  type  as  well. 

There  is  a  considerable  increase  in  the  large  mononuclear  percentage.  Nucleated 
reds  and  myelocytes  are  invariably  absent.  It  must  be  remembered  that  we  have  a 
group  of  cases  showing  splenomegaly  which  are  syphilitic  in  origin  and  which,  as  a 
rule,  give  a  positive  Wassermann.  Clinically  or  haematologically  they  resemble  true 


SPLENIC  ANAEMIA  241 

Banti's  disease  but  pathologically  the  spleen  shows  a  fibrosis  instead  of  the  marked 
increase  in  lymphatic  tissue  characteristic  of  Banti's  disease. 

In  the  tropical  splenomegaly  or  kala-azar  we  have  a  marked  leukopenia  with 
a  marked  reduction  in  the  percentage  of  polymorphonuclears.  The  Gaucher  type 
of  splenic  anaemia  does  not  show  as  pronounced  and  early  an  anaemia  as  in  Banti's 
type. 

Certain  conditions  which  partly  resemble  myelogenous  leukaemia 
and  partly  pernicious  anaemia  are  designated  leukanaemia.  Some  con- 
sider this  to  belong  to  the  group  of  diseases  in  which  the  multiple 
myeloma  is  placed. 

In  splenomegalic  polycythaemia  we  have  a  red  count  of  from  9,000,000  to  10,000,000. 
The  Hb.  percentage  may  be  200.  There  is  also  a  leukocytosis  up  to  50,000.  Pa- 
tients are  cyanosed  and  have  a  very  large  spleen. 

Splenic  anaemia  of  infancy  usually  occurs  between  the  ages  of  twelve  and  twenty- 
four  months.  The  spleen  is  notably  enlarged  and  in  many  cases  the  liver  is  equally 
so.  The  red  cells  are  not  greatly  diminished  in  number,  2,500,000  to  3,000,000 
being  usual  findings.  Nucleated  reds  are  abundant.  While  a  leukocytosis  of 
30,000  to  50,000  is  often  present  it  is  markedly  less  than  that  of  splenomyelogenous 
leukaemia  and  the  increase  in  white  cells  is  more  of  those  of  lymphocyte  type. 

The  color  index  is  very  low. 

Another  splenomegaly  of  children,  clinically  resembling  kala-azar,  is  caused  by 
Leishmania  iufantum. 


16 


PART  III 
ANIMAL  PAR ASITO LOGY 

CHAPTER  XV 

GENERAL  CONSIDERATIONS  OF  CLASSIFICATION  AND 

METHODS 

ANIMALS  that  are  in  all  respects  alike  we  term  a  Species.  Of  course 
the  male  and  female  of  a  species  may  be  very  unlike,  but  as  a  result  of 
mating  they  produce  young  having  characteristics  similar  to  the 
parents.  Now,  if,  as  in  the  case  of  the  mosquitoes  causing  yellow  fever, 
we  find  some  with  straight  silvery  lines  and  others  uniformly  showing 
crescentic  silvery  bands  about  thorax,  yet  resembling  each  other  closely 
in  the  respect  of  being  dark,  brilliantly  marked  mosquitoes,  we  should 
consider  them  as  being  separate  species  with  a  certain  relationship 
to  which  the  term  Genus  is  applied. 

The  term  "genus"  is  of  wider  application  than  the  word  "species."  Thus 
animals  which  agree  in  the  main  characteristics  of  size,  proportion  of  parts,  and 
general  structure  are  placed  in  the  same  genus. 

In  naming  a  species  we  always  first  write  the  name  of  the  genus 
which  has  a  Greek  or  Latin  name,  commencing  with  a  capital,  and 
follow  with  the  specific  name,  which  latter  commences  with  a  small 
letter.  Thus  we  designate  the  dark  silver-marked  mosquitoes  as 
belonging  to  the  genus  Stegomyia;  those  showing  the  characteris- 
tics of  curved  silver  bands  and  two  central  parallel  lines  (lyre  pat- 
tern) on  dorsal  surface  of  thorax  we  designate  as  Stegomyia  calopus; 
the  species  with  only  the  straight  silver  lines  we  call  Stegomyia 
scutellaris. 

The  specific  name  may  be  a  noun  in  the  genitive.  If  an  adjective  it  must  agree  in 
gender  with  the  generic  name. 

It  is  permissible  to  have  a  masculine  noun  as  a  specific  name  with  a  feminine 
generic  name. 

243 


244         CONSIDERATIONS   OF   CLASSIFICATION  AND   METHODS 

If  the  specific  name  is  a  modern  patronymic  we  add  i  in  the  case  of  a  man  or  ae  for 
a  woman  to  the  exact  and  complete  name  of  the  person. 

Again,  certain  genera  show  resemblances  which  enable  us  to  make  broader 
groupings  to  which  we  apply  the  term  Subfamily.  Thus  the  genus  Stegomyia 
and  the  genus  Culex  have  the  similar  characteristics  of  palpi  in  the  female  being 
shorter  than  the  straight  proboscis;  we  therefore  classify  all  species  of  Stegomyia 
and  all  species  of  Cidex  under  the  designation  Culicinae.  The  name  of  a  subfamily 
ends  in  "inae."  Now,  again,  certain  insects  are  different  from  others  in  having 
scales  on  the  wings.  We  find  that  not  only  do  the  Culicinse  have  such  character- 
istics, but  the  same  is  observed  with  the  Anophelinae  and  other  similar  scale-wing 
insects.  All  of  these  we  term  a  Family  and  we  speak  of  the  Culicidae,  meaning 
the  family  of  mosquitoes.  The  name  of  a  family  ends  in  "idae."  Many  families 
are  not  subdivided  into  subfamilies,  but  are  directly  separated  into  genera.  Again, 
a  genus  may  have  only  a  single  species. 

At  times  a  family  may  be  raised  to  superfamily  rank — the  sub- 
families then  becoming  families.  Thus  the  families  Ixodidae  and 
Argasidae  belong  to  the  superfamily  Ixodoidea.  The  termination  for  a 
superfamily  is  "oidea". 

When  there  are  a  number  of  families  agreeing  closely  in  some  striking  character- 
istic, we  group  them  together  into  an  Order;  thus,  the  family  of  mosquitoes  closely 
resembling  many  other  families  of  insects  in  possessing  a  pair  of  well-developed 
wings  are  grouped  in  the  order  Diptera;  all  of  which  resemble  certain  other  animals 
in  the  possession  of  a  distinct  head,  thorax  and  abdomen  with  three  pairs  of  legs 
projecting  from  the  thorax.  This  collection  of  animals  we  call  a  Class;  thus,  we 
speak  of  the  class  Insecta.  It  will  be  observed  that  the  insects  have  no  internal 
skeleton,  but  instead  a  chitinous  cuticle,  the  exoskeleton.  Spiders,  ticks,  etc., 
resemble  them  in  this  respect,  and  we  now  apply  to  all  such  animals  the  wider 
designation,  Branch  or  Phylum  Arthropoda. 

Inasmuch  as  the  animal  kingdom  is  divided  into  the  branches  Protozoa,  Pori- 
fera,  Ccelenterata,  Echinodermata,  Vermes,  Arthropoda,  Mollusca  and  Chordata, 
we  see  that  the  branch  is  the  largest  grouping  we  employ.  To  descend  in  the 
scale  we  have  belonging  to  the  branch,  the  classes;  to  the  class,  the  orders;  to 
the  order,  the  families;  to  the  family,  the  subfamilies;  to  the  subfamily,  the 
genera;  to  the  genus,  the  species.  Occasionally  a  species  is  further  divided  into 
subspecies. 

By  a  type  species  we  understand  the  species  of  a  genus  always  re- 
ferred to  as  representing  the  genus. 

While  other  species  of  a  genus  may  for  good  reason  be  transferred  to  another 
genus  the  type  species  is  permanently  in  the  genus.  Many  favor  alliteration  for 
type  species,  as  Heterophyes  heterophyes.  When  a  species  is  transferred  to  a  new 
genus  the  specific  name  goes  with  it. 

The  male  animal  is  designated  by  the  sign  of  Mars  (d"),  the  female  by  that  of 
Venus  (9). 


KEY  TO   ANIMAL  PARASITES 


245 


CLASSIFICATION  OF  PHYLA  OF  IMPORTANCE  IN  ANIMAL  PARASITOLOGY 
(According  to  Stiles) 

1.  Unicellular  animals,  as  the  parasites  of  malaria Protozoa. 

Pluricellular  animals;  metazoa 2 

2.  Body  more  or  less  flattened  dorsoventrally 4 

Body  ordinarily  round  in  transverse  section 

3.  Body  never  annulated,  never  provided  with  legs;  no  jaws  present 5 

Body  annulated,  or  at  least  provided  with  mouth  parts;  usually  breathe  through 

a  tracheal  system;  adults  with  jointed  legs 7 

4.  Intestine,  but  no  anus,  present;  one  or  two  suckers  present;  body  not  seg- 

mented; parasitic  in  liver,  lungs,  blood,  intestine;  occasionally  elsewhere; 
flukes Trematoda. 

Intestine  absent;  two  or  four  suckers  on  head;  body  of  adults  segmented;  tissue 
usually  contains  calcareous  corpuscles;  adults  (tapeworms)  parasitic  in  intes- 
tine; larvae  (bladder  worms)  parasitic  elsewhere Cestoda. 

Intestine  and  anus  present;  ventral  sucker  on  posterior  end;  body  annulated  like 
an  earthworm;  parasitic  in  upper  air-passages,  or  externally,  leeches,  blood- 
suckers   Hirudinei. 

5.  Intestine  absent;  armed  rostellum  present;  very  rare  in  man,  in  intestine,  thorn- 

headed  worms Acanthocephali. 

Intestine  present;  no  armed  rostellum 6 

6.  Intestine  rudimentary  in  adult;  rare,  accidental  parasites  in  intestine  of  man,  hair 

snakes  or  horse-hair  worms Gordiacea. 

Intestine  present;  parasitic  in  intestine,  muscles,  lymphatics,  etc.,  very  common 
and  important;  roundworms Nematoda. 

7.  Six  legs  present  in  adult;  wings  present  in  most  species;  larvae  annulated  much  like 

an  earthworm;  breathe  through  tracheae;  adults  ectoparasites;  occasionally 
larva  is  parasitic  under  skin,  or  in  wounds,  or  an  accidental  parasite  in  the 
intestine;  insects Insecta. 

Eight  legs  present  in  adult,  six  legs  in  larva;  head  and  abdomen  coalesced; 
ectoparasites;  some  burrow  under  the  skin  or  live  in  the  hair  follicles; 
acarines Acarina. 

Four  claws  around  the  mouth;  larva  encysted  in  various  organs;  adult  occasion- 
ally parasitic  in  nasal  passages;  tongue  worms Linguatulidae. 

Numerous  legs  present;  occasionally  accidental  parasites  in  nasal  passages  or 
intestine,  thousand  leggers Myriapoda. 

There  are  certain  terms  employed  in  animal  parasitology  which  it  is 
necessary  to  understand.     Among  these  we  shall  refer  to  the  following: 

1.  True  Parasitism. — By  this  is  understood  the  condition  where  the  parasite  does 
harm  to  the  host,  deriving  all  the  benefit  of  the  association.     A  good  example  of  this 
would  be  the  hookworm  infecting  man  or  animals. 

2.  Mutualism. — In  such  an  association  there  is  mutual  benefit  to  each  party  of  the 
association.     An  instance  of  this  would  be  the  presence  of  colon  bacilli  in  the  intes- 
tines.    The  bacillus  is  furnished  a  suitable  habitat  and  in  return  protects  its  host 
against  strictly  pathogenic  bacteria. 


246         CONSIDERATIONS    OF   CLASSIFICATION   AND   METHODS 


Another  example  would  be  the  oyster  crab  found  inside  the  oyster  shell. 

3.  Commensalism. — Here  there  is  benefit  to  the  parasite,  but  no  injury  to  the 
host.     An  example  of  this  kind  would  be  furnished  in  the  case  of  the  Trichomonas 
vaginalis  which  lives  in  the  vaginal  mucus,  but  so  far  as  known,  does  no  injury  to  the 
host. 

If  the  Entamceba  coll  be  nonpathogenic  this  would  be  another  example. 

4.  Nomenclature. — When  the  thousands  of  different  species,  genera, 
etc.,  of  animals  are  considered,  it  will  be  readily  perceived  that,  unless 
some  system   existed  for   their   designation,   indescribable  confusion 
would  prevail.     To  avoid  this,  the  International  Code,  based  on  the 
rules  of  Linnaeus  (tenth  edition  of  Systema  naturae,  1758,  is  basis  of 
binary  zoological  nomenclature) ,  requires  Latin  or  Latinized  names. 

In  printed  matter  the  zoological  name  should  be  in  italics,  that  of  the  family  in 
Roman  type.  The  name  of  the  author  of  a  specific  name  is  written  immediately  after 
the  name  without  punctuation  and  may  be  followed  by  the  year  of  publication  set  off 
by  a  comma,  thus:  Ascarls  lumbricoldes  Linnaeus,  1758.  Should  the  name  of  the 
author  appear  in  parentheses  it  indicates  that  he  proposed  the  specific  name  but 
placed  the  species  in  another  genus  than  that  in  which  it  now  appears,  and  the  name 
of  the  author  responsible  for  placing  the  species  in  the  present  genus  may  be  written 
after  the  name  of  the  original  author  of  the  species;  for  example,  Davainea  mada- 
gascariensis  (Davaine,  1869)  Blanchard,  1891,  tells  us  that  Davaine  proposed  the 
specific  name  madagascarlensls  in  1869  but  placed  it  in  some  other  genus  and  that 
Blanchard  in  1891  transferred  it  to  the  genus  Davainea.  There  are  certain  rules 
governing  the  naming  of  animals.  Of  these,  the  law  of  priority  provides  that  the  oldest 
published  name,  under  the  code,  of  any  genus  or  species  is  its  proper  zoological  name. 
The  history  of  the  naming  of  the  organism  of  syphilis  illustrates  this  well.  Schau- 
dinn  gave  this  organism  in  1905  the  name  of  Splroch&ta  pallida.  Ehrenburg,  in 
1838,  had  used  the  name  Splrochata  for  animals  of  a  different  character,  so  that  this 
designation  of  the  genus  was  not  permissible  under  the  code.  Villemin,  a  little  later, 
proposed  the  generic  name  Splronema.  This  term,  however,  was  found  to  have 
been  used  in  1864  by  Meek  for  a  genus  of  molluscs  and  by  Klebs  in  1892  for  a  genus 
of  flagellates.  Consequently,  being  a  homonym,  it  was  not  available. 

(A  generic  name  can  be  applied  to  only  one  animal  genus  and  if  a  similar  name  is 
subsequently  given  another  genus  it  is  a  homonym  and  is  to  be  rejected.) 

On  December  2,  1905  Stiles  and  Pfender  then  proposed  the  name  Micros pironema, 
but  as  Schaudinn  published  on  Oct.  26,  1905  the  designation  Treponema,  the 
name  Treponema  pallldum  had  to  be  accepted  as  the  proper  zoological  name  for  the 
organism  of  syphilis. 

Of  unusual  interest  is  the  question  of  the  name  of  the  old-world  hookworm. 
Dubini,  in  1843,  named  a  nematode  found  by  him  in  man  Agchylostoma.  By  the 
law  of  priority  this  spelling  would  have  been  the  correct  one  had  he  not  stated  in  a 
footnote  that  the  generic  name  was  derived  from  two  Greek  words  cryxuXoer  and 
or6/Lta.  Having  indicated  the  origin  of  the  name  it  became  subject  to  the  rules  for 
correct  transliteration,  which  is  Ancylostoma. 


ZOOLOGICAL  NOMENCLATURE  247 

In  case  of  larva  and  adult  or  male  and  female,  formerly  considered 
different  animals  but  subsequently  found  to  be  the  same,  the  oldest 
available  name  becomes  the  name  of  the  species. 

Another  point  is  that  names  are  not  definitions,  consequently  the  fact  of  lack  of 
appropriateness  of  any  name  is  no  objection  to  its  continuation.  This  will  appeal 
to  anyone  as  a  wise  provision,  because  if  a  different  name  were  substituted  each  time 
a  designation  more  descriptive  or  applicable  was  invented  it  would  be  utterly  destruc- 
tive to  system.  When  it  is  considered  that  some  of  our  parasites  have  approximately 
fifty  different  designations,  for  the  most  part  given  by  medical  observers,  it  will  be 
appreciated  how  much  the  zoologist  has  aided  us  in  trying  to  eliminate  all  but  the 
single  proper  zoological  name. 

It  is  a  rule  of  zoological  nomenclature  that  zoological  names  are 
independent  of  botanical  ones  so  that  the  prior  use  of  a  generic  name 
for  a  plant  is  not  an  objection  to  its  use  for  an  animal. 

The  objections  so  frequently  heard  among  physicians  in  connection  with  adopting 
new  names  for  old  ones  are  not  well  founded.  Wherever  confusion  has  reigned,  the 
establishment  of  order  always  results  in  temporary  greater  confusion.  There 
is  no  doubt  that  the  student  taking  up  this  subject  a  few  years  hence  will  have  the 
satisfaction,  thanks  to  the  zoologist,  of  only  having  to  burden  his  mind  with  one  name 
for  one  parasite. 

There  is  only  one  correct 'name  for  an  animal  and  all  other  names 
are  synonyms. 

The  principal  cause  of  changes  of  names  is  that  our  conception  of  the  relationships 
of  animals  changes. 

5.  Terminology. — This  applies  to  appropriate  designations  for  different  organs, 
symptoms,  etc.,  and  is  not  subject  to  any  rule  other  than  that  of  good  usage. 

Thus  the  terms  cirrus  in  the  case  of  the  male  copulatory  organ  of  flukes,  spicule 
for  the  same  in  nematodes  and  penis  in  connection  with  insects  would  be  instances 
of  terminology. 

6.  Pseudoparasitism. — Where  organisms  enter  the  body  accidentally  and  when 
such  sojourn  in  the  body  of  man  plays  no  part  in  the  life  history  of  the  organism 
we  employ  the  term  pseudoparasitism.     For  example:  Fly  larvae  swallowed  by 
man  and  passed  out  in  the  faeces.     We  also  use  the  terms  temporary  parasites 
(bedbug)  and  permanent  parasites  (liver  fluke). 

7.  Hosts. — The  animal  in  which  a  parasite  undergoes  its  sexual  life  is  called  the 
definitive  or  final  host,  that  in  which  it  passes  its  larval  existence  the  intermediary 
host.     For  example:  Man  is  the  intermediary  host  of  the  malarial  parasite,  the 
mosquito  the  definitive  host.     A  single  animal  may,  however,  be  both  definitive 
and  intermediary  host;  thus  Trichinella  may  pass  its  larval  existence  in  the  muscles 
of  man  and  its  sexual  life  in  his  intestines. 

8.  Heredity,  Congenitalism.— Hereditary   characteristics  are  those  which  were 
present  in  the  ovum  or  spermatozoon  before  fertilization;  congenital  ones  those 
which  occur  after  fertilization.     South  African  tick  fever  is  probably  an  instance 


248         CONSIDERATIONS    OF   CLASSIFICATION   AND   METHODS 


of  heredity,  the  spirochaetes  having  been  found  in  the  ovary  and  ova  of  the  fema 
tick. 

9.  Heterogenesis,  Parthenogenesis. — Offspring  differs  from  parent,  but  after 
one  or  more  generations  there  is  reversion  to  the  parent  form. 

Strictly  speaking  the  term  heterogony  applies  to  reproduction  when  a  sexual 
generation  alternates  with  a  parthenogenetic  one.  Where  a  nonsexual  genera- 
tion, as  by  division  or  budding,  alternates  with  a  sexual  one  the  process  is  called 
metagenesis.  In  parthenogenesis  reproduction  eggs  develop  without  the  occurrence 
of  fertilization  by  spermatozoa. 

In  coccidiosis  we  have  a  sexual  cycle  (sporogony)  alternating  with  a  nonsexual 
one  (schizogony) .  In  the  infection  with  Strongyloides  we  have  a  sexual  cycle 
alternating  with  a  parthenogenetic  one.  In  malaria  we  have  a  sexual  generation, 
a  nonsexual  one  and  according  to  Schaudinn,  a  parthenogenetic  one,  which  latter 
accounts  for  malarial  relapses. 

10.  Homology   and  Analogy. — By   homology   we   understand    the   anatomical 
correspondence  of  the  organ  of  one  animal  to  that  of  another.     Thus  the  foreleg  of 
a  quadruped  and  the  wing  of  a  bird  are  homologous  organs.     Analogy  refers  to 
physiological  or  functional  agreement,  thus  the  lungs  of  mammals  and  gills  of  fish, 
both  with  respiratory  functions,  are  analogous  organs.     The  first  trace  or  appear- 
ance of  an  organ  in  an  embryo  is  known  as  the  anlage  of  the  organ. 

11.  Protista. — Haeckel  proposed  this  name  for  unicellular  animals  and  plants, 
thus  including  protozoans  and  protophytes  in  a  kingdom  separate  from  the  animal 
and  vegetable  kingdoms. 

We  have  sufficient  difficulty  in  drawing  the  line  between  an  animal  and  vegetable 
organism  so  that  to  make  a  demarcation  of  a  new  kingdom  from  the  two  usually 
recognized  would  add  to  our  difficulties. 


nale 


CHAPTER  XVI 


Class 


THE  PROTOZOA 

CLASSIFICATION  OF  PROTOZOA 
Order  Genus 


Rhizopoda 

(Sarcodina) 
These   throw  out   protoplas- 
mic projections  called  pseudo- 
podia. 


Gymnamceba 


Flagellata 

(Mastigophora) 
These    move    by    means    of 
undulating       membranes       or 
flagella. 


Infusoria 

(Ciliata) 

These  have  contractile  vacu- 
oles  and  numerous  fine  cilia 
which  are  shorter  than  flagella 
and  have  a  sweeping  stroke. 

Sporozoa 

These  have  no  motile  organs. 
They  live  parasitically  in  the 
cells  or  tissues  of  other  animals. 
Reproduction  by  spores. 


Entamoeba 


Leydenia 


Spirochaeta 

Schizotrypanum 
Treponema 

Trypanosoma 

Trichomonas 

Tetramitus 

Lamblia 

Babesia 

Leishmania 


Heterotricha        Balantidium 


Coccidiaria 


Eimeria 
Isospora 


Haemosporidia      Plasmodium 


249 


Species 
E.  coli 

E.  histolytica 
E.  tetragena 
E.  gingivalis 


L.  gemmipara 

S.  recurrentis 
S.  vincenti 
{  S.  duttoni 
S.  carter! 
S.  refringens 
S.  cruzi 
T.  pallidum 
T.  pertenue 
T.  gambiense 
T.  rhodesiense 
T.  vaginalis 
T.  intestinalis 
T.  mesnili. 
L.  intestinalis 
B.  bigemina 
L.  donovani 
L.  tropica 
L.  infantum 
B.  coli 


E.  stiedae 
I.  bigemina 

P.  vivax 
P.  malariae 
P.  falciparum 


250  THE   PROTOZOA 

NOTE. — Hartmann  and  others  have  grouped  the  Haemosporozoa  and  the  Haemo- 
flagellata  in  an  order  BINUCLEATA.  The  main  characteristic  is  the  possession  of 
two  differentiated  nuclei,  the  kinetonucleus  and  the  trophonucleus,  at  some  develop- 
mental or  transitional  stage.  While  trypanosomes  plainly  show  these  characteristics 
certain  others,  as  the  malarial  parasites  and  the  leishman-donovan  bodies,  having 
been  modified  as  the  result  of  cell  parasitism,  do  not  do  so.  This  grouping  together 
of  the  blood  flagellates  and  sporozoa  under  the  name  Binucleata  has  been  considered 
by  many  protozoologists  as  possibly  convenient  but  not  resting  on  sufficient  ground 
to  cause  organisms  with  similar  life  histories  as  Plasmodium  and  Coccidium  to  be 
separated  and  the  former  to  be  placed  with  the  blood  flagellates  in  a  new  grouping. 

THE  PROTOZOA 

By  the  term  protozoa  we  understand  a  branch  of  animals  in  which  a 
single  cell  is  morphologically  and  functionally  complete;  it  is  not  one  of 
a  number  of  cells  going  to  make  up  a  complex  individual  and  dependent 
on  such  a  combination  as  is  the  case  with  the  metazoa  (there  is  no 
differentiation  into  tissues  in  protozoa). 

Recognizing  the  fact  that  certain  protozoa  have  characteristics  which  make 
it  impossible  to  draw  a  distinction  between  them  and  plants  Haeckel  has  proposed  the 
name  Protista  as  a  designation  for  all  simple  and  primitive  living  organisms  whether 
they  be  plants  or  animals.  In  such  a  classification  we  would  have  the  kingdom  of 
Protista  as  well  as  the  animal  and  vegetable  kingdoms.  In  such  a  grouping  the  bac- 
teria would  be  the  lower  types  and  the  fungi  and  protozoal  organisms  the  higher  ones. 

The  protozoal  cells  are  made  up  of  protoplasm  which  is  divided  into  nucleus  and 
cytoplasm.  The  cytoplasm  is  at  times  separated  into  an  external,  hyaline  portion, 
the  ectoplasm  or  ectosarc  and  an  internal  granular  portion,  the  endoplasm  or  endo- 
sarc.  The  functions  of  the  ectosarc  are  protective,  locomotor,  excretory  and  sensory; 
those  of  the  endosarc  trophic  and  reproductive.  Protozoa  may  be  holozoic  (animal 
like)  or  holophytic  (plant  like),  saprophytic  (fungus  like),  or  parasitic  (living  at  the 
expense  of  some  other  animal  or  plant). 

The  nucleus  is  characterized  by  concentration  of  the  so-called  chromatin  sub- 
stance of  the  cell.  This  chromatin  however  is  usually  combined  with  achromatin. 
The  usually  accepted  test  for  chromatin  is  the  staining  affinity  for  basic  aniline  dyes. 
This  test  is  now  known  to  be  unsatisfactory  as  other  substances  than  chromatin  may 
stain  even  more  intensely.  When  chromatin  is  scattered  through  the  cytoplasm,  as 
extranuclear  aggregations,  such  chromatin  granules  are  called  chromidia.  There  are 
cells  where  the  chromidia  take  the  place  of  the  nucleus  and  from  which  a  nucleus 
may  be  formed.  Chromidia  may  arise  from  nuclei  and  nuclei  from  chromidia.  The 
nucleus  is  made  up  of  a  network  of  linin  in  which  achromatic  reticulum  is  contained 
the  nuclear  sap  or  karyolymph.  As  a  rule  an  achromatic  nuclear  membrane,  con- 
tinuous with  the  reticulum,  separates  the  nucleus  from  the  cytoplasm.  In  addition 
we  have  a  substance  which  is  achromatic  (plastin)  and  which  is  the  imbedding  sub- 
stance for  chromatin  grains.  These  plastin  chromatin  combinations  are  called 
karyosomes.  The  nucleoli  are  probably  pure  plastin.  Plastin  is  to  be  regarded  as  a 


RHIZOPODS  251 

secretion  or  modification  of  chro matin  made  to  serve  as  a  matrix  for  the  chromatin. 
Chromatin  may  be  concentrated  in  a  single  mass  so  that  the  nuclear  space  looks 
like  a  vesicle  with  a  central  chromatin  mass  (vesicular  nucleus)  or  numerous  chro- 
matin grains  may  be  scattered  through  the  nuclear  space  (granular  nucleus).  The 
centrosome,  which  presides  over  cell  division,  is  usually  located  just  outside  the 
nucleus.  In  some  protozoa  however  the  centrosome  is  within  the  nucleus  and 
is  often  seen  inside  of  a  karyosome  and  is  then  called  a  centriole.  The  centrosome 
may  also  function  over  kinetic  activities  (flagellar  motion)  and  is  then  termed 
blepharoplast. 

When  appearing  as  a  small  granule  at  the  base  of  the  flagellar  apparatus  it  is  called 
the  basal  granule.  When  there  are  extensions  from  it  to  the  nucleus  we  have 
rhizoplasts. 

Certain  protozoa,  as  trypanosomes,  show  a  differentiation  of  nuclei,  the  larger 
trophonucleus  governing  the  functions  of  general  metabolism  and  the  smaller  kine- 
tonucleus  directing  the  motor  activities.  Infusoria  have  a  larger  macronucleus 
which  contains  vegetative  chromatin  and  a  smaller  micronucleus  which  contains 
reserve  reproductive  chromatin. 

Reproduction  of  protozoa  may  be  by  fission,  when  the  nucleus  and  cytoplasm 
divide  into  two  by  simple  division. 

When  the  nuclei  divide  into  a  number  of  daughter  nuclei,  which  is  followed  by 
multiple  division  of  the  cytoplasm,  we  have  sporulation. 

Instead  of  fission  we  may  have  sexual  reproduction  or  conjugation  (zygosis). 
Here  the  nuclei  of  the  separate  sexual  individuals  (gametes)  are  termed  pronuclei  and 
the  product  of  their  fusion  a  synkaryon. 

Where  a  single  cell  has  division  of  its  nucleus  with  subsequent  fusion  of  these 
daughter  nuclei  to  form  a  synkaryon  the  process  is  termed  autogamy. 

If  two  similar  cells  conjugate  the  term  is  isogamy;  if  dissimilar  as  the  macroga- 
metes  and  microgametes  of  malaria,  anisogamy. 

The  process  of  sexual  union  is  termed  syngamy  and  is  of  two  kinds  (i)  when  the 
two  gametes  fuse  completely  or  copulation  and  (2)  when  they  remain  separate 
and  only  exchange  nuclear  material  or  conjugation. 

The  structures  of  protozoa  concerned  in  movement,  metabolism,  etc.,  are  termed 
organelles.  Of  the  former,  pseudopodia,  flagella,  cilia  and  myonemes  (contractile 
fibrils  which  give  support  to  the  body  cell  of  certain  protozoa)  may  be  given  and  food 
vacuoles  and  contractile  vacuoles  of  the  latter.  The  contractile  vacuole  which  is 
probably  an  excretory  organelle  is  absent  in  almost  all  parasitic  protozoa.  It  is 
however  present  in  ciliates. 

RHIZOPODA  (SARCODINA) 

In  this  class  of  protozoa  the  pseudopodia  serve  the  double  purpose 
of  nutrition  and  locomotion.  These  protoplasmic  extensions  may  be 
quite  broad  or  very  narrow — the  lobose  and  the  reticulose. 

As  a  rule,  the  thicker  the  pseudopod  the  more  rapid  the  movement.  Some  rhizo- 
pods  have  hard  shell-like  coverings  which  are  secreted  in  or  on  the  ectosarc.  These 
skeletons  have  openings  through  which  the  pseudopods  project.  The  pseudopodia 
may  be  made  up  only  of  ectoplasm  or  both  ectoplasm  and  endoplasm  may  take  part. 


252  THE   PROTOZOA 

Amoeboid  movement  always  starts  in  the  ectoplasm.  In  addition  to  the  nucleus, 
which  the  so-called  chromatin-staining  methods  of  Romanowsky  bring  out  as  reddish 
areas,  or  black  with  iron  haematoxylin,  we  frequently  observe  aggregations  of  chro- 
matin-staining  material  in  the  cytoplasm.  These  cytoplasmic  chromatin  bodies 
(chromidial  bodies)  are  of  importance  in  differentiating  the  encysted  pathogenic 
amoeba  from  the  nonpathogenic  one.  This  extranuclear  chromatin  is  supposed  to 
play  a  part  in  the  more  intricate  divisions  which  such  protozoa  undergo.  Food 
vacuoles  and  contractile  vacuoles  are  present  in  many  rhizopods. 


INTESTINAL  AMCEB^E 

There  are  certainly  two  species  of  intestinal  amoebae  having  man 
for  a  host,  the  one  pathogenic,  Entamceba  histolytica  and  the  other  a 
harmless  commensal,  Entamoeba  coli. 

Schaudinn,  in  1903,  described  the  pathogenic  amoeba,  which  he  named  E.  histolyt- 
ica, as  follows:  i.  Distinct,  highly  refractile  and  tenacious  ectoplasm.  He  consid- 
ered this  tough  external  portion  of  the  cytoplasm  as  the  explanation  of  the  ability 
of  the  pathogenic  amoeba  to  bore  its  way  into  the  intestinal  submucosa.  2.  Eccen- 
tric nucleus  which  was  indistinct  by  reason  of  little  chromatin.  3.  Reproduction  by 
peripheral  budding  in  which  small  aggregations  of  chromatin  reached  the  periphery 
of  the  cytoplasm  and,  enclosed  in  a  resistant  capsule,  broke  off  from  the  parent 
amoeba  and  constituted  the  infecting  stage. 

For  the  nonpathogenic  E.  coli  he  noted,  i.  No  distinction  between  a  granular 
endoplasm  and  refractile  ectoplasm.  2.  Centrally  placed  and  sharply  outlined 
nucleus,  rich  in  chromatin  and  3.  Encystment  with  the  formation  of  eight  nuclei, 
which  nuclei  or  amcebulae  form  the  infecting  stage. 

The  pseudopodia  of  E.  histolytica  are  actively  projected  as  long  finger-like  processes 
which  show  the  ectoplasm  quite  distinctly,  while  the  pseudopodia  of  E.  coli,  are 
lobose  and  sluggishly  projected  and  show  a  uniformly  opaque  grayish  color. 

In  1907  Viereck  and  later  Hartmann  recognized  a  pathogenic 
amoeba  with  four  nuclei  in  its  encysted  form,  to  which  was  given  the 
name  E.  tetragena. 

All  authorities  now  consider  that  Schaudinn  made  an  error  in  observation  as  to 
the  existence  of  peripheral  budding  for  E.  histolytica,  so  that  we  recognize  but  two 
types  of  encystment,  one  with  a  larger  cyst  and  thicker  cyst  wall,  with  eight  nuclei 
and  an  absence  of  chromidial  bodies — E.  coli  and  the  other,  smaller,  with  a  thin  cyst 
wall,  four  nuclei  and  chromidial  bodies  in  the  encysted  stage,  the  pathogenic  amoeba, 
E.  histolytica.  Synonym.  E.  tetragena. 

In  the  vegetative  stage  the  human  amoebae  are  best  differentiated  by  the  nuclear 
structure.  In  E.  coli  the  nucleus  is  vesicular  with  a  thick  nuclear  membrane  and 
the  chromatin  chiefly  deposited  on  the  undersurface  of  the  nuclear  membrane. 
In  haematoxylin-stained  specimens  this  chromatin  often  seems  deposited  in  quadrant 
aggregations. 

Whitmore  noted  a  broad  clear  zone  as  surrounding  the  karyosome  of  the  tetra- 


THE    AMCEB.E   OF   MAN 


253 


gena  nucleus  of  E.  histolytica,  a  distinction  from  the  central  karyosome  of  E.  coll 
which  has  only  a  very  narrow  clear  zone  surrounding  it. 

At  the  present  we  differentiate  between  two  types  of  nucleus— the 
histolytica  one,  which  is  found  in  acute  attacks  of  dysentery  and 


0 


FIG.  59. — Important  pathogenic  protozoa  of  the' intestinal  tract.  (la)  Motile  E, 
coli.  Note  large  amount  and  peripheral  arrangement  of  chromatin  in  nucleus.  ( ib ) 
Encysted  E.  coli.  Note  larger  size  than  E.  histolytica  cyst,  8  ring  form  nuclei  and 
absence  of  chromidial  bodies.  (2)  Motile  E.  histolytica  from  acute  dysenteric  stool. 
Note  histolytica  nucleus  with  scanty  chromatin.  (3)  Tetragena  type  of  E.  histo- 
lytica from  case  of  chronic  dysentery.  Note  greater  amount  of  chromatin  and  central 
karyosome  with  centriole.  (4a)  Preencysted  E.  histolytica  from  carrier.  Note  small 
size  and  heavy  peripheral  ring  of  chromatin  in  nucleus  making  this  feature  of  chrom- 
atin in  nucleus  similar  to  the  larger  E.  coli.  (4_b)  Encysted  E.  histolytica  from 
dysentery  convalescent.  Note  small  size,  4  ring  nuclei  and  a  dark  chromatin  stain- 
ing mass,  "chromidial  body."  (sa  and  5b)  Motile  and  encysted  cultural  amoebae 
from  Manila  water  supply.  (6a  and  6b)  Oocyst  and  sporozoite  production  in  4 
spores  of  Eimeria  stiedae.  (ya  and  76)  Oocyst  with  2  sporoblasts  and  oocyst  with 
2  spores  containing  4  sporozoites  of  Isopora  bigemina.  (8a  and  8b)  Vegetative  and 
encysted  Trichomonas  intestinalis .  (pa  and  pb)  Vegetative  and  encysted  Lamblia 
intestinalis.  (10)  Balantidium  coli.  Illustrations  of  amoebae  from  Walker — others 
from  Doflein. 

shows  a  delicate  nuclear  membrane  with  scattered  chromatin  granules 
in  its  inner  side  and  as  a  rule  a  very  small  central  karyosome.  The 
tetragena  type  has  a  much  thicker  nuclear  membrane  and  is  the  one 
generally  found  in  pathogenic  amoebae  in  chronic  dysentery.  Of 


254  THE   PROTOZOA 


not; 


course  instead  of  the  vegetative  nucleus  of  tetragena  type  one  may 
in  chronic  cases  or  in  convalescence  come  across  the  four  nuclei  cysts 
of  E.  tetragena. 

Animal  experimentation  upon  kittens  with  E.  coli  by  Schaudinn,  Craig  and 
Wenyon  have  been  unsuccessful  as  to  production  of  dysenteric  manifestations.  On 
the  other  hand  all  of  these  experimenters  produced  typical  lesions  and  dysenteric 
manifestations  in  kittens  injected  rectally  or  fed  with  material  containing  patho- 
genic amoebae. 

Wenyon  produced  a  liver  abscess  in  one  of  his  experiments.  In  man  the  dislodg- 
ment  of  amoebae  containing  material  from  amoebic  intestinal  ulcerations  and  the 
plugging  of  the  portal  capillaries  by  such  emboli  gives  us  the  starting-point  of  a  liver 
abscess.  The  exciting  cause  is  Entamceba  histolytica  which  in  the  liver  continues 
the  same  production  of  a  gelatinous  necrosis  as  is  carried  on  in  the  submucosa  of  the 
large  intestine  or  appendix. 

Darling  has  been  so  successful  in  his  experimental  work  with  kittens  that  he 
compares  the  colon  of  a  kitten  to  a  test-tube  and  suggests  the  procedure  of  rectal 
injections  of  material  containing  amoebae  as  a  means  of  differentiating  the  two  human 
amoebae. 

On  the  other  hand  Walker  was  unable  to  infect  kittens  and  monkeys  with  material 
containing  pathogenic  amoebae  and  he  makes  the  statement  that  such  failures  would 
indicate  the  greater  susceptibility  of  man  to  infection,  as  he  was  able  to  infect  17 
out  of  20  men  with  one  feeding  of  such  material. 

Recently  Walker  and  Sellards  have  published  a  most  important 
paper. 

The  experiments  were  made  in  men  who  had  been  under  observation  for  years 
at  Bilibid  Prison,  whose  food  was  cooked  and  the  water  they  drank  distilled.  More- 
over, there  were  complete  records  of  examination  for  intestinal  parasites,  including 
entamcebae.  They  were  under  complete  control  and  the  existence  or  possibility  of 
natural  infection  with  amoebae  was  reduced  to  a  minimum.  All  the  men  fed  patho- 
genic amoebae  were  volunteers  and  each  signed,  in  his  native  dialect,  an  agreement  to 
the  conditions  of  the  experiment. 

The  first  series  of  experiments  was  with  cultural  amoebae,  in  order  to  refute  state- 
ments that  amoebae  cultivated  from  water  or  other  nonparasitic  sources,  as  well 
as  from  dysenteric  stools,  are  capable  of  living  in  man  parasitically  or  of  producing 
dysenteric  symptoms.  Twenty  feeding  experiments  were  made  by  Walker  and 
Sellards  with  cultures  of  amoebae  without  the  development  in  a  single  instance  of 
dysentery  or  the  finding  of  such  amoebae  in  the  stools  upon  microscopical  examina- 
tion. In  13  cases  they  recovered  the  amoebae  in  cultures  from  the  feces  from  the 
first  to  the  sixth  day,  but  never  afterward.  They  stated  definitely  that  cultural 
amoebae  are  nonpathogenic. 

The  next  experiments  were  with  Entamoeba  coli.  In  the  20  cases  fed  with  material 
containing  Entamaba  coli  there  was  a  uniform  failure  to  recover  them  culturally  and 
in  no  instance  was  dysentery  produced.  Seventeen  became  parasitized  as  the  result 
of  a  single  feeding  in  from  one  to  eleven  days,  the  entamcebae  being  found  in  the 
stools  and  persisting  in  their  appearance  in  the  stools  for  extended  periods.  They 


EPIDEMIOLOGY  OF  AMCEBIASIS  255 

concluded  that  Entamosba  coli  is  an  obligate  parasite,  nonpathogenic,  and  cannot  be 
cultured. 

The  third  series  of  20  feedings,  carried  on  by  Walker  alone,  was  with  Entamceba 
histolytica.  The  material  was  mixed  with  powdered  starch  or  magnesium  oxide  and 
given  in  gelatin  capsules.  In  these  experiments  they  obtained  tetragena  cysts  in 
the  stools  of  men  fed  only  motile  Entamceba  histolytica,  and  motile  Entamosba  histolyt- 
ica in  the  stools  of  men  who  were  fed  only  tetragena  cysts  and,  finally,  an  alterna- 
tion of  motile  E.  histolytica  and  tetragena  cysts  in  the  stools  of  a  man  having  a 
recurrent  attack  of  amoebic  dysentery. 

Seventeen  of  the  men  became  parasitized  after  the  first  feeding;  one  required  three 
feedings,  and  two,  who  did  not  become  parasitized  at  the  first  feeding,  were  held  as 
controls.  The  average  time  for  parasitization  was  nine  days.  Only  four  of  the  18 
parasitized  men  developed  dysentery,  which  came  on  after  20,  57,  87,  and  95  days, 
respectively,  after  the  ingestion  of  the  infecting  material. 

In  four  cases  fed  with  material  from  acute  dysenteric  stools  or  from  amoebae  con- 
taining pus  from  liver  abscess,  and  containing  motile  amcebae,  there  was  no  resulting 
dysentery,  the  four  cases  of  experimental  dysentery  resulting  from  feeding  of  material 
from  normal  stools  of  carriers. 

As  regards  the  cases  which  became  parasitized,  but  did  not  develop  dysentery, 
it  is  suggested  that  the  amoebae  live  as  commensals  in  the  intestine  of  the  host  and 
only  penetrate  the  intestinal  mucosa  and  become  tissue  parasites  when  there  occurs 
depression  of  the  natural  resistance  of  the  host  or  as  the  result  of  some  lesion  of  the 
intestine.  That  the  pathogenic  amcebae  are  more  than  harmless  commensals,  how- 
ever, is  shown  by  the  fact  that  they  alone,  and  not  the  nonpathogenic  Entamosba 
coli,  are  capable  of  penetrating  a  possibly  damaged  intestinal  mucosa. 

Epidemiology. — The  old  idea  that  water,  fruit  or  vegetables,  from 
which  one  can  isolate  amcebae  upon  culture,  are  sources  of  infection 
must  be  abandoned,  as  such  cultural  amcebae  are  known  to  have  no 
pathogenic  relation  to  man. 

The  chief  factor  in  the  spread  of  amoebic  dysentery  would  seem  to  be  the  encysted 
amcebse  in  the  stools  of  convalescents  or  healthy  carriers  rather  than  the  motile  ones 
in  dysenteric  stools.  This  probably  explains  the  endemic  rather  than  epidemic 
characteristics  of  the  spread  of  amoebic  dysentery  because  if  the  innumerable  vege- 
tative amcebse  in  dysenteric  stools  were  equally  operative  with  the  more  sparsely 
eliminated  cysts  there  would  be  epidemics  of  amoebic  dysentery  similar  to  those  of 
bacillary  dysentery. 

Our  present  view  is  that  the  carrier  is  the  chief  factor  in  the  spread 
of  amoebic  dysentery  and  when  such  an  individual  has  to  do  with  the 
preparation  of  food  he  becomes  a  particular  source  of  danger. 

Vegetative  amcebze  undergo  disintegration  in  a  short  time  after  the  stool  is  passed 
so  that  they  are  probably  rarely  concerned  in  amoebic  infections  but  the  resisting 
cysts  may  be  washed  from  a  dried  stool  into  a  water  supply  or  even  be  transported  in 
dust  to  lodge  on  unprotected  food  stuffs. 


256  THE   PROTOZOA 


Flies  may  possibly  act  as  transmitting  agents. 

We  can  as  a  rule  differentiate  bacillary  from  amoebic  dysentery  by  the  more 
sudden  and  acute  onset  of  the  former  together  with  fever  and  other  evidences  of 
toxaemia. 

Again  the  number  of  stools  in  bacillary  dysentery  is  usually  greater  and  the  amount 
of  each  stool  less  in  quantity.  The  stool  of  bacillary  dysentery  is  of  a  milky  white- 
ness from  the  large  number  of  pus  cells,  while  that  of  amoebic  dysentery  is  more 
viscid  and  tinged  with  disintegrated  blood  giving  it  a  grayish  green  or  brown  color. 
The  mucopurulent  mass  in  bacillary  dysentery  may  be  flecked  or  streaked  with 
blood.  The  therapeutic  results  following  emetine  injections  are  of  value  in  diagnosis. 
Gangrenous  types  of  dysentery  are  similar  whether  due  to  bacillary  or  amoebic 
infection.  Chronic  dysentery  of  bacillary  origin  is  much  like  amoebic  dysentery 
clinically. 

Laboratory  Diagnosis. — In  the  fresh  specimen  of  the  milky  muco- 
purulent mass  of  bacillary  dysentery  one  observes  large  numbers  of 
pus  cells  and  particularly  very  large  phagocytic  cells  which  greatly 
resemble  amoebae.  Upon  staining  with  Gram's  stain  one  may  find 
numerous  Gram-negative  bacilli  in  the  cystoplasm  of  the  cell. 

The  large  cells  which  resemble  amoebae  are  often  vacuolated,  thus  intensifying 
the  similarity.  They  are  nonmotile,  however,  and  do  not  show  the  small  ring 
nucleus  which  is  so  characteristic  of  the  vegetative  human  amoebae.  The  nucleus 
of  the  confusing  cells  is  also  larger,  approximating  one-fourth  the  size  of  the  cell. 

For  bringing  out  the  nuclear  characteristics  of  human  amoebae  Walker  recom- 
mends fixation  of  thin  moist  smears  in  sublimate  alcohol  (absolute  alcohol  i  part, 
sat.  aq.  sol.  bichloride  2  parts)  for  ten  to  fifteen  minutes.  These  smears  are  then 
well  washed  with  water  and  stained  with  alum  haematoxylin  for  five  minutes.  The 
nuclear  characteristics  are  noted  under  etiology.  With  vegetative  amoebae  I  have 
obtained  beautiful  results  with  vital  staining  which  can  best  be  done  by  tinging  the 
faeces  emulsion  with  a  i%  aqueous  solution  of  neutral  red.  I  have  also  had  good 
results  by  emulsifying  the  faeces  in  a  drop  of  i  or  2%  formalin  and  then  adding  a 
drop  of  2%  acetic  acid.  The  mixture  is  then  tinged  with  either  neutral  red  or 
methyl  green. 

For  distinguishing  the  encysted  form  of  Entamceba  coli  one  can  obtain  beautiful 
results  by  emulsifying  the  faeces  in  Gram's  iodine  solution.  Owing  to  the  glycogenic 
reaction  given  by  E.  coli,  the  round  amoebae,  with  its  eight  nuclei  stands  out 
very  distinctly. 

For  diagnosing  the  four  nucleated  cyst  of  the  pathogenic  amoeba  one 
gets  better  results  with  haematoxylin  as  this  brings  out  not  only  the  four 
nuclei  but  the  chromidial  bodies  as  well.  The  use  of  the  iodine  solu- 
tion is,  however,  almost  as  satisfactory  for  pathogenic  cysts  as  for 
nonpathogenic  ones.  Again  where  cysts  are  not  abundant  one  has 
little  success  in  finding  them  in  iron  haematoxylin  preparations  which 


DIFFERENTIATION  OF  AMCEBAE 


257 


staining  I  prefer  to  alum  haematoxylin.  It  was  formerly  customary 
to  recommend  the  administration  of  salts  prior  to  examining  for 
amoebae. 

Walker  warns  that  such  a  procedure  gives  us  amoebae  which  are  difficult  to  differ- 
entiate, the  nuclear  characteristics  of  E.  coli  and  the  tetragena  nucleus  of  E.  histo- 
lytica  being  much  alike  as  they  both  contain  much  chromatin.  In  a  dysenteric 
stool  the  histolytica  type  of  nucleus,  containing  but  little  chromatin,  does  not 
resemble  the  nucleus  of  E.  coli. 

He  prefers  the  examination  of  formed  stools  obtained  without  a 
purgative. 

Walker  also  notes  the  advantages  of  examining  a  specimen  with  a  %-inch  ob- 
jective as  encysted  amoebae  are  easily  picked  up.  In  opposition  to  the  usual  recom- 
mendation of  text-books  to  report  only  on  motile  amcebae,  he  recommends  the 
making  of  a  differential  diagnosis  on  nonmotile  encysted  forms.  This  however  is 
now  generally  accepted  by  experienced  workers  as  true.  The  four  nuclei  cysts  of 
E.  histolytica  are  from  n  to  14  microns  in  diameter  while  the  eight  nuclei  ones  of  E. 
coli  are  from  1 6  to  25  microns  in  diameter. 

Sellards  and  Baetjer  note  that  inoculation  of  kittens  per  rectum  or 
by  feeding  dysenteric  stools  rich  in  amcebae  has  resulted  in  infection 
in  about  50%  of  experiments. 

By  inoculating  the  material  directly  into  the  caecum  they  were 
able  to  infect  every  one  of  their  kittens.  They  were  also  able  to 
propagate  a  strain  of  amcebae  through  a  series  of  animals  for  several 
months. 

The  intracaecal  inoculations  yielded  positive  results  in  diagnosis  of 
human  amcebiasis  when  the  clinical  manifestations  were  obscure 
and  the  amcebae  in  the  discharges  so  few  and  atypical  as  to  make  such 
an  examination  unsatisfactory. 

As  differentiating  the  two  entamcebae  Walker  gives  the  following  table: 


MOTILE  STAGE 
A.  Entamceba  histolvtica 


B.  Entamceba  coli 


1.  Appearance  hyaline. 

2.  Refractiveness  more  feeble. 

3.  Movements  active  in  the  fresh  stool. 

4.  Nucleus  more  or  less  indistinct. 

5.  Chromatin  of  nucleus  scanty. 

17 


1.  Appearance  porcelaneous. 

2.  Refractiveness  more  pronounced. 

3.  Movements  sluggish. 

4.  Nucleus  distinct. 

5.  Chromatin  of  nucleus  abundant. 


258 


THE   PROTOZOA 


ENCYSTED  STAGE 
A.  Entamceba  histolytica 


B.  Entamoeba  coli 


1.  Cyst  smaller. 

2.  Cyst  less  refractive. 

3.  Cyst  usually  contains  elongated  re- 
fractive bodies  known  as  "chromidial 
bodies." 

4.  Nuclei  never  more  than  four. 

5.  Cyst  wall  thinner. 


1.  Cyst  larger. 

2.  Cyst  more  refractive. 

3.  Cysts    do  not    contain    "chromidial 
bodies." 

4.  Nuclei  eight,  occasionally  more. 

5.  Cyst  wall  thicker. 


A  method  for  bringing  out  the  nuclear  features  is  as  follows:  take  a  loopful  of 
2%  acetic  acid  and  a  loopful  of  2%  formalin.  Tinge  the  mixture  to  a  rose  color 
with  neutral  red  and  then  stir  in  a  little  saturated  aqueous  solution  methyl  green, 
using  a  toothpick  which  has  been  dipped  into  the  methyl  green. 

In  staining  with  iron  haematoxylin  or  better  with  phosphotungstic  haematoxylin 
proper  fixation  is  very  important.  Fix  in  100  parts  of  sat.  aq.  sol.  bichloride  to 
which  is  added  50  c.c.  absolute  alcohol  and  5  drops  glacial  acetic  acid.  The  stain 
should  be  poured  on  the  moist  smear  of  faeces.  The  fixative  should  be  heated  to 
6o°C.  and  should  only  act  for  ten  to  twenty  seconds.  Then  place  in  cold  sub- 
limate alcohol  for  ten  minutes  wash  in  70%  alcohol  colored  to  a  rich  port  wine 
color  with  iodine,  then  in  70%  alcohol,  then  in  water  and  then  stain  as  preferred. 
Some  like  a  carmine  stain.  The  smears  should  be  moist  when  fixed  with  bichloride 
fixatives.  A  small  loopful  of  Meyer's  albumin  fixative  to  emulsify  is  a  great  aid. 

AMCEB.E  or  PYORRHOEA  ALVEOLARIS 

Recently  much  importance  has  been  attached  to  certain  amoebae 
found  deep  in  the  pus  pockets  of  affected  teeth.  They  are  best  ob- 
tained after  wiping  away  the  superficial  pus  and  then  scraping  material 
from  the  depths  of  the  pockets  with  a  wooden  toothpick.  They  may 
be  emulsified  in  salt  solution  but  saliva  is  better  for  the  obtaining  of 
motility.  The  name  is  Entamceba  gingivalis  and  it  is  probably  the  one 
described  under  the  names  E.  buccalis  and  E.  dentalis.  It  rather  re- 
sembles E.  histolytica  in  having  a  greenish  refractile  ectosarc  and  an 
indistinct  nucleus.  Encysted  forms  have  not  been  observed. 

It  is  open  to  question  whether  these  amoebae  are  the  cause  or  whether  various 
streptococci  bring  about  the  condition.  A  symbiotic  relationship  may  be  opera- 
tive. Emetine  seems  often  of  value  especially  when  combined  with  an  autogenous 
vaccine.  The  recent  enthusiasm  for  emetine  in  treatment  of  pyorrhoea  seems  to  be 
disappearing. 

Entamceba  gingivalis  varies  from  10  to  25/1  in  diameter.  The  nucleus  is  much 
smaller  than  those  of  the  intestinal  amoebae  and  has  a  distinct  nuclear  membrane  en- 
closing a  deeply  stained  karysome.  On  the  whole  it  is  poor  in  chromatin,  in  this 
respect  rather  resembling  the  histolytica  nucleus. 


SPIROCH^TES  259 

Pseudopod  projection  is  less  active  than  that  of  E.  histolytica  but  more  so  than  E. 
coli.  There  is  no  distinction  between  endoplasm  and  ectoplasm  and  the  nucleus  is 
indistinct.  Encysted  forms  are  very  rarely  seen  and  these  do  not  show  evidence  of 
reproduction. 

FLAGELLATA  (MASTIGOPHORA) 

In  this  class  of  protozoa  the  adults  have  flagella  for  the  purposes  of 
locomotion  and  the  obtaining  of  food. 

Some  flagellates  more  or  less  resemble  rhizopods  in  being  amoeboid  and  in  having 
an  ectoplasm  and  an  endoplasm.  The  body  is  frequently  covered  by  a  cuticle 
(periplast).  Some  flagellates  have  a  definite  mouth  part,  the  cytostome,  which  leads 
to  a  blind  oesophagus;  others  absorb  food  directly  through  the  body  wall.  In  addi- 
tion to  flagella,  some  flagellates  possess  an  undulating  membrane.  All  flagellates 
possess  a  nucleus  and  some  have  contractile  vacuoles.  The  flagellum  may  arise 
directly  from  the  nucleus  or  from  a  small  kinetic  nucleus,  the  blepharoplast  (micro- 
nucleus  or  basal  granule). 

The  most  important  flagellates  of  man  are  the  hsemoflagellates.  Among  these 
we  may  include  the  blood  spirochsetes  and  the  organism  of  syphilis,  which  have 
many  resemblances  to  the  spiral  forms  of  bacteria,  together  with  the  three  genera  in 
which  protozoal  characteristics  are  marked,  namely,  Leishmania,  Trypanosoma 
and  Trypanoplasma.  In  addition  we  have  flagellates  in  the  intestinal  canal  and  in 
the  vaginal  secretion.  Some  authors  place  the  genus  Piroplasma  with  the  flagellates 
and  there  has  been  controversy  concerning  the  nature  of  certain  projections  from 
these  bodies.  It  would  seem  preferable,  however,  to  consider  them  under  the 
Sporozoa. 

Spirochaeta 

The  generic  term  Spirochata  is  applied  to  flagellates  having  a  spiral 
shape,  an  undulating  membrane,  and  no  flagella.  This  genus  is  one 
about  which  there  are  two  views:  one,  that  the  members  belong  to  the 
bacteria;  the  other,  that  they  are  protozoa.  The  absence  of  demon- 
strable nucleus  and  blepharoplast  makes  them  apparently  vegetable  in 
nature  while  the  variations  in  thickness,  the  fact  of  transmission  by  an 
arthropod,  and  indications  of  a  longitudinal,  rather  than  a  transverse 
division,  would  indicate  protozoal  affinities. 

It  would  seem  from  recent  investigations  that  both  methods  occur— longitudinal 
division  occurring  when  there  are  few  organisms  in  the  blood  and  transverse  at  the 
height  of  the  infection. 

Minchin  has  adopted  the  name  Spiroschaudinnia,  proposed  by  Sambon,  for  the 
parasitic  blood  spirochaetes. 

Spirochsetes  of  Relapsing  Fevers.— Relapsing  fevers  are  caused  by 
organisms  generally  considered  as  protozoal  in  their  nature  and  be- 
longing to  the  flagellates. 


260  THE   PROTOZOA 


The  generic  name  Spiroschaudinnia  is  preferred  by  some  to  the  more  commonly 
accepted  Spirochata.  East  and  West  African  relapsing  fevers,  or  tick  fever,  is 
caused  by  S.  duttoni  and  the  transmission  is  through  the  bite  of  an  argasine  tick, 
Ornithodorus  moubata.  Not  only  does  the  tick  itself  become  infected  by  the  taking  in 
of  blood  containing  spirochaetes  but  likewise  transmits  the  infection  to  its  progeny. 
Leishman  considers  that  when  the  spirochaetes  are  taken  into  the  alimentary  tract 
of  the  tick  there  is  a  breaking  up  of  the  spirochaetes  into  small  granules  which  reach 
the  Malpighian  tubules.  They  also  invade  the  ovary  and  the  ova.  It  was  thought 
that  these  granules  were  the  infecting  agents  and  that  they  were  excreted  in  the 
fluid  of  the  coxal  glands  or  passed  out  with  the  faeces.  More  recently  it  has  been 
claimed  that  these  granules  have  no  relation  to  the  infection,  which  is  due  to  spiro- 
chaetes as  such.  At  any  rate  this  infection  of  man  seems  to  be  by  the  contamination 
method,  the  material  from  faeces  and  coxal  glands  being  rubbed  into  the  wound  made 
by  the  tick  bite.  The  ticks  hide  in  the  cracks  about  the  old  native  huts  and  bite  the 
sleeping  inmates.  There  may  be  quite  a  local  reaction  at  the  site  of  the  bite. 


nly 


FIG.  60. — Spirochaetae  of  relapsing  fever  from  blood  of  a  man.     (Kolle  and 

Wassermann.) 

Spircchata  duttoni  has  been  cultured  by  Noguchi,  by  utilizing  his  methods  for 
culturing  the  organism  of  syphilis.  In  such  cultures  he  has  noted  longitudinal 
division  rather  than  transverse,  this  fact  rather  favoring  a  protozoal  as  against  a 
bacterial  nature.  This  spirochaete  is  from  24-30  microns  long,  about  0.45  microns 
broad  and  has  a  corkscrew  motility.  It  is  readily  transmissible  to  a  number  of 
laboratory  animals,  as  white  rats,  etc.  The  spirochaete  of  northern  African  re- 
lapsing fever,  S.  Berbera  causes  the  disease  as  seen  in  north  Africa  and  Egypt.  It 
is  transmitted  by  lice,  Nicolle  and  others  having  shown  that  the  spirochaetes  make 
their  way  from  the  alimentary  tract  to  the  body  cavity  of  the  louse.  They  have 
shown  that  the  bite  alone  of  an  infected  louse  is  innocuous  and  also  that  the  faeces 
are  noninfective,  when  injected  into  monkeys.  Emulsions  of  infected  lice,  however, 
when  rubbed  into  wounds,  produce  the  disease  in  monkeys. 

It  is  by  crushing  the  louse,  by  scratching  or  otherwise,  that  the 
spirochaetes  contained  in  the  ccelomic  fluid  reach  and  penetrate  the 


SYPHILIS  26l 

wound  of  the  bite.     This  is  therefore  a  contaminative  method  of 
infection. 

Mackie  has  shown  that  the  Indian  relapsing  fever,  which  is  caused  by  S.  carteri,  is 
probably  transmitted  by  the  louse,  and  it  is  probable  that  the  conditions  under  which 
the  infection  takes  place  are  similar  to  those  occurring  with  S.  berbera  infections. 
With  the  European  relapsing  fever,  bedbugs  have  been  suggested  as  transmitting 
agents.  The  probabilities  however  are  that  this  infection  is  caused  by  lice. 

A  relapsing  fever  of  Persia  is  transmitted  by  a  tick  of  the  genus  Ornithodorus. 
There  is  great  variation  in  the  description  of  the  different  spirochjetes,  and  fre- 
quently measurements  are  given  for  short  forms  and  long  forms.  They  also  vary 
from  wave-like  lines  to  corkscrew  spirals.  Again,  different  species  have  different 
types  and  different  activities  of  movement.  As  a  rule  they  are  about  20  X  0.4 
microns.  Noguchi  has  recently  cultivated  the  various  species  of  pathogenic  human 
spirochaetes  by  employing  a  method  similar  to  that  used  in  cultivating  the  organism 
of  syphilis.  He  noted  longitudinal  division  in  his  cultures. 

S.  vincenti. — This  is  a  very  delicate  spiral-shaped  organism  which  has  been  found 
in  conjunction  with  a  fusiform  bacillus  in  a  throat  inflammation,  usually  termed 
Vincent's  angina. 

S.  refringens. — This  Spirochata  is  frequently  associated  with  the  Treponema 
pallidum  and  is  common  in  genital  ulcerations.  It  is  thicker,  has  less  regular  and 
more  flattened  curves  and  stains  more  readily.  By  "dark  ground  illumination"  it 
is  thicker,  of  a  yellow  tint  instead  of  pure  white,  and  moves  in  its  entire  length. 

i 

Treponema 

The  genus  Treponema  has  no  undulating  membrane  and  has  a 
flagellum  at  each  end. 

Treponema  pallidum  (Spirochseta  pallida). — This  is  the  cause  of 
syphilis.  It  was  discovered  by  Schaudinn  and  Hoffmann  in  1905  in 
various  syphilitic  lesions  and  was  cultured  by  Noguchi  in  1912.  It  is 
characterized  by  the  very  geometric  regularity  of  the  spirals,  which  are 
deeply  cut,  and  in  focusing  up  and  down  continue  in  focus  (like  a 
corkscrew).  They  require  about  thirty  minutes  to  stain  distinctly 
with  Giemsa's  stain  and  the  attenuated  ends  or  flagella  should  always 
be  noted  before  reporting  their  presence. 

Treponemata  are  found  in  the  cellular  areas  surrounding  the  thickened  blood- 
vessels and  in  the  coats  of  the  larger  arteries.  To  stain  them  in  section  Levaditi's 
method  is  the  best. 

The  India-ink  method  of  Burri  is  highly  recommended.  Take  one  loopful  of 
secretion  from  a  chancre  and  deposit  it  on  one  end  of  a  slide.  Emulsify  in  this  a  small 
quantity  of  Gunther  and  Wagner's  ink  such  as  can  be  obtained  by  touching  the  end 
of  a  new  spatulate  toothpick  in  the  ink.  Use  the  toothpick  for  mixing.  I  use  the 
method  of  sticking  a  fine  glass  pipette  into  the  underlying  corium  of  the  chancre  and 


262  THE   PROTOZOA 

get  serum  in  that  way  or  by  squeezing  the  chancre  afterward.  Mix  and  make  a 
smear  as  for  blood.  When  dry  examine  with  the  oil  immersion  objective  and  the 
treponemata  will  be  found  to  stand  out  as  white  spirals  against  a  dark  background. 
Treponemata  often  appear  as  if  bent  in  the  middle. 

Harrison  prefers  collargol  to  india  ink.  One  part  of  collargol  is  put  in  a  bottle 
with  19  parts  of  water  and  well  shaken.  This  shaking  is  repeated.  One  loopful 
of  the  suspected  serum  and  one  loopful  of  the  collargol  suspension  are  mixed  and 
smeared  out  and  examined  as  for  the  india-ink  method. 

Cultivation. — T.  pallidum  has  been  cultivated  anaerobically  in  horse  serum  by 
Schereschewsky.  The  cultures  contained  other  organisms.  Muhlens,  by  growing 
anaerobically  on  horse-serum  agar  (i  to  3),  claims  to  have  obtained  pure  cultures. 
Animal  inoculations  with  this  material  were  negative,  however. 

Noguchi  has  cultivated  T.  pallidum  under  strict  anaerobic  conditions  in  a  medium 
of  ascitic  fluid  containing  a  piece  of  fresh  sterile  tissue,  preferably  placenta.  The 
growth  is  faintly  hazy  and  does  not  have  an  offensive  odor.  Spirochceta  micro- 
dentium  shows  similar  morphology  but  the  cultures  have  a  foul  odor.  Sp.  macro- 
dentium  is  similar  culturally  but  differs  morphologically. 

Luetin. — When  cultures  of  T.  pallidum,  grown  for  one  or  more  weeks  in  ascitic 
fluid  agar  and  ascitic  fluid  are  ground  in  a  mortar,  heated  to  6o°C.  for  one  hour 
then,  with  the  final  addition  of  i%  trikresol,  we  have  an  emulsion  called  "luetin." 
This  extract  produces  an  allergic  reaction  on  the  skin  of  certain  syphilitics  (luetin 
reaction).  To  carry  out  the  test  luetin  is  introduced  intradermally  at  the  insertion 
of  the  left  deltoid  and  a  control  emulsion  of  agar  media  injected  in  the  right  arm. 
A  negative  result  shows  as  an  erythema  without  pain  or  papule  formation.  Positive 
reactions  show  as  papules  vesicles  or  even  pustules  giving  rise  to  discomfort  for 
several  days.  Not  only  do  we  have  papular  and  pustular  type  reactions  but  also 
torpid  ones  (taking  ten  days  or  more  to  develop).  While  the  control  side  usually 
becomes  normal  in  forty-eight  hours  yet  in  latent  and  tertiary  syphilis  the  control 
may  show  almost  as  marked  a  reaction.  The  term  "Umstimmung"  is  applied  to 
this  susceptibility  to  trauma  of  the  skin  of  those  having  tertiary  syphilis.  Some 
cases  of  parasyphilitic  infections  which  are  negative  to  the  Wassermann  test  give 
a  positive  luetin  reaction.  Tertiary  yaws  cases  frequently  give  a  positive  luetin 
reaction.  See  comparison  of  Wassermann  and  luetin  statistics. 

Noguchi  has  recently  demonstrated  T.  pallidum  in  all  layers  of  the  cerebral 
cortex  except  the  outermost  one  in  12  cases  out  of  70  cases  of  general  paresis 
examined. 

Diagnosis. — In  diagnosis  either  use  the  dark  ground  illuminator  or  make  a  thin 
smear  from  the  sanious  oozing  after  vigorous  friction  of  the  chancre  with  gauze, 
taking  up  this  blood-stained  serum  on  the  end  of  a  slide  and  smearing  the  surface 
of  a  second  slide  with  the  adhering  material.  It  is  in  most  cases  more  satisfactory 
to  curet  the  lesion,  in  this  way  obtaining  material  from  the  areas  of  the  thickened 
arteries.  Fontana's  silver  staining  method  is  an  excellent  one. 

In  the  diagnosis  of  cerebrospinal  syphilis  we  use,  in  addition  to  the  Wassermann 
test  of  the  blood,  (i)  the  Nonne-Apelt  reaction  in  which  about  i  c.c.  of  a  saturated 
aqueous  solution  of  ammon.  sulphate  is  added  to  an  equal  amount  of  cerebrospinal 
fluid.  If  turbidity  or  rather  opalescence  appear  immediately,  or  within  three 
minutes,  the  test  is  positive.  We  now  use  a  ring  test.  (2)  The  counting  of  the 
lymphocytes  in  the  cerebrospinal  fluid.  A  lymphocytosis  occurs  in  cerebrospinal 


YAWS 


263 


syphilis,  tabes  and  general  paresis.  (3)  The  Wassermann  test,  using  the  cerebro- 
spmal  fluid  instead  of  blood-serum.  (4)  The  colloidal  gold  test  of  Lange  These 
various  examinations  of  spinal  fluid  are  taken  up  in  detail  in  chapter  on  cytodiagnosis 
and  spinal  fluid  examinations. 

T.  pertenue.— An  organism  of  similar  morphology  was  first  reported 
by  Castellani  as  present  in  yaws.  It  is  found  in  smears  and  sections  as 
with  T.  pallidum. 


Q 


:."  .. 


8 


9 


§, 

eC 


FIG.  61. — Binucleata,  (Haemoflagellata  and  Haemosporozoa).  i.  Schizotrypanum 
cruzij  (a)  Merozoite  just  entering  r.b.c.;  (b)  fully  developed  trypanosome  form  in 
blood;  (c)  form  found  in  intestine  Conor  hinus;  {d)  form  in  salivary  gland  of  Conor hi- 
nus;  (e)  merocyte  from  the  schizogenous  cycle  in  lungs.  2.  Leishmania  donovani; 
Parasites  from  spleen  smear,  free  and  packed  in  phagocytic  cell;  (b)  and  (c)  flagel- 
late forms  from  cultures.  3.  Trypanosoma  gambiense.  4.  Plasmodium  vivax;  (a) 
young  schizont;  (b)  uninfected  red  cell;  (c)  red  cell,  punctate  basophilia;  (d)  merocyte; 
(e)  macrogamete;  (/")  adult  schizont.  5.  Plasmodium  malaria;  (a)  half-grown 
schizont  showing  equatorial  band;  (b)  macrogamete;  (c)  merocyte;  (d)  young 
schizonts.  6.  Plasmodium  falciparum;  (a)  red  cell  showing  multiple  infection;  (b) 
young  ring  from;  (c)  crescent;  (d)  young  schizont  on  periphery  of  r.b.c.  7.  (a) 
Treponema  pallidum;  (b)  Spirochceta  refringens.  8.  Treponema  pertenue. 

A  point  of  distinction  between  these  spirochaetes  is  that  the  T.  pallidum  is  found 
in  abundance  in  sections  from  a  chancre  about  the  thickened  arteries  in  the  corium, 
while  in  sections  from  a  yaws  nodule  the  T.  pertenue  is  found  chiefly  in  the  region 
of  the  interpapillary  pegs  of  the  Malpighian  layer  of  the  epidermis  where  they  bound 
the  papillary  layer  of  the  corium. 


264  THE   PROTOZOA 


T.  pertenue  has  been  cultivated  in  the  same  way  as  T.  pallidum  and  Nichols 
infected  rabbits  by  intratesticular  injection.     A  disease  of  Guam  known  as  gangosa 
is  possibly  connected  with  a  tertiary  form  of  yaws.     In  persons  who  have  had  yaws 
a  positive  Wassermann  reaction  seems  to  be  given  in  a  higher  percentage  than  is 
true  for  syphilis.     Salvarsan  is  also  more  specific  for  yaws  than  for  syphilis. 

TRYPANOSOMES  OF  SLEEPING  SICKNESS 

The  African  trypanosomiases  follow  infection  with  two  species  of 
trypanosomes;  the  more  virulent  type  of  the  disease,  occurring  in 
South  Central  Africa,  being  due  to  Trypanosoma  rhodesiense,  trans- 
mitted by  Glossina  morsitans  and  that  of  less  severe  type,  but  of 
more  general  distribution,  being  due  to  T.  gambiense  and  transmitted 
by  Glossina  palpalis.  The  very  important  Trypanosoma  brucei,  which 
is  the  devastating  agent  in  the  African  horse,  dog  and  cattle  disease, 
nagana,  is  also  transmitted  by  Glossina  morsitans  and  there  exists 
the  opinion  that  this  trypanosome  is  identical  with  T.  rhodesiense. 

These  trypanosomes  are  blood  flagellates  and  are  typical  of  the  Binucleata  in 
possessing  two  chromatin  staining  areas,  the  larger  and  more  centrally  situated 
mass  being  the  tropho  or  macronucleus  and  the  smaller,  but  more  deeply  staining  one, 
the  kineto  or  micronucleus  (Blepharoplast).  Trypanosomes  have  a  fusiform  or 
fish-shaped  body  which  stains  blue.  Near  the  less  pointed,  nonflagellated  end, 
usually  called  the  posterior  end,  is  the  deeply  stained  blepharoplast.  Behind  this 
is  a  vacuole  and,  taking  origin  from  this  part  of  the  trypanosome,  is  the  flagellum. 
This  borders  an  undulating  membrane  attached  to  the  body  and  then,  carried  along 
to  the  other  extremity,  projects  free  as  a  long,  whip-like  flagellum. 

In  fresh  preparations  the  body  of  the  trypanosome  progresses  in  the  direction  of 
its  flagellated  end,  although  occasionally  it  will  be  observed  to  move  in  the  opposite 
direction. 

T.  gambiense  varies  much  in  length  and  breadth.  The  normal  type,  as  found  in 
the  blood,  varies  from  14  to  20  microns,  while  longer  forms,  20  to  24  microns,  are 
growth  ones  and,  in  the  longest  ones  (23  to  33  microns),  we  have  those  preparing 
to  divide  longitudinally.  The  normal  short  forms  are  the  ones  from  which  the 
development  takes  place  in  the  tsetse  fly.  In  width  these  flagellates  are  from  1.5  to 
2  microns.  The  blepharoplast  is  oval  and  the  nucleus  situated  about  the  center. 

With  T.  rhodesiense  the  nucleus  is  typically  located  almost  adjacent 
to  the  blepharoplast.  As  a  matter  of  fact  it  may  require  the  passage  of 
this  trypanosome  through  rats  to  bring  out  these  "posterior  nuclear 
forms,"  the  nuclear  location  being  at  times  almost  entirely  that  of 
T.  gambiense.  In  addition  to  the  characteristics  of  nucleus  being  near 
the  blepharoplast,  this  trypanosome  is  more  virulent  for  laboratory 
animals  than  T.  gambiense,  agreeing  in  this  respect  with  the  more  severe 
clinical  course  in  man. 


has 


SLEEPING   SICKNESS  265 

When  the  tsetse  fly,  Glossina  palpalis,  feeds  on  a  man  in  whose  peripheral  circu- 
lation there  are  normal  type  trypanosomes  we  have  an  accumulation  of  such  forms  in 
the  middle  and  posterior  portions  of  the  gut.  From  the  eighth  to  the  eighteenth  day 
long,  slender  forms  develop  and  pass  forward  into  the  pro  ventriculus.  None  of  the  in- 
testinal forms  can  cause  infection  when  injected  into  animals.  These  proventricular 
types  work  their  way  into  the  salivary  ducts  and  thence  into  the  salivary  glands, 
where  further  development  takes  place.  Here  we  have  shorter  forms  developing, 
which  are  similar  in  morphology  to  the  normal  blood  type.  It  is  at  this  stage  that 
the  fly  becomes  infective  by  the  passing  of  these  trypanosomes  down  the  salivary 
ducts  and  through  the  channel  in  the  hypopharynx  to  the  subcutaneous  tissues  of 
the  person  bitten.  High  temperatures,  75  to  8s°F.,  are  favorable  to  development, 
while  low  temperatures,  60  to  7o°F.,  are  inimical  to  development,  but  do  not  kill 
the  ingested  trypanosomes.  This  explains  the  long  period  which  at  times  elapses 
before  a  fly  becomes  infective.  Under  favorable  conditions  a  fly  becomes  infective 
in  twenty  to  twenty-four  days  and  remains  infective  the  rest  of  its  life,  up  to  185  days. 
The  infection  is  not  transmitted  to  the  pupa.  This  is  an  inoculative,  cyclical  or 
indirect  type  of  infection.  It  is  usually  considered  that  a  tsetse  fly  whose  proboscis 
has  just  been  contaminated  with  trypanosome  blood  is  capable  of  transferring  the 
infection  for  a  few  hours.  This  would  be  a  mechanical  or  direct  method  of  infection 
and  such  power  for  infection  only  lasts  for  a  few  hours. 

When  tsetse  flies  feed  on  animals  infected  with  trypanosomes  only 
from  2  to  6%  become  infective.  Again,  it  has  been  shown  that  where 
the  wild  animals  on  which  tsetse  flies  feed  may  show  an  infection  of 
from  16  to  50%  yet  not  more  than  two  out  of  every  1000  tsetse  flies, 
caught  and  tried  out  on  susceptible  animals,  show  themselves  infective. 

Both  of  the  human  trypanosomes  of  Africa  have  been  cultured  by  using  the 
N.N.N.  medium  in  which  rat's  blood  was  substituted  for  that  of  the  rabbit.  Human 
blood  will  also  serve  as  a  substitute.  Growth  however  is  not  constant. 

For  the  laboratory  diagnosis  we  may  use  peripheral  blood  with  some 
thick  film  method.  The  examination  of  preparations  from  the  per- 
ipheral blood  is  usually  very  discouraging.  Very  much  better  results 
(in  fact  some  prefer  this  method  to  any  other)  can  be  obtained  by  tak- 
ing 10  to  20  c.c.  of  blood  into  about  25  c.c.  of  citrated  salt  solution, 
centrifuging  two  or  three  times  and  examining  the  sediment  of  the 
third  centrifugalization.  Button  and  Todd  prefer  to  centrifuge 
citrated  blood  and  to  collect  the  leukocyte  layer  for  examination  as  is 
done  in  opsonic  work. 

The  English  workers  usually  prefer  the  gland  puncture  method,  using  a  sterile 
but  dry  hypodermic  needle.  Water  in  the  needle  distorts  both  leishman  bodies  and 
trypanosomes. 

In  the  sleeping  sickness  stage  trypanosomes  can  almost  constantly  be  found  in  the 
cerebrospinal  fluid. 


266  THE    PROTOZOA 


Some  prefer  to  inoculate  susceptible  animals,  particularly  the  guinea-pig  or 
monkey,  with  blood  or  gland  juice  from  the  suspected  case.  A  very  satisfactory 
material  is  an  emulsion  from  an  excised  gland  which  may  be  inoculated  intraperi- 
toneally  into  white  rats.  The  further  course,  after  animal  inoculation,  is  the  ex- 
amination of  the  blood  of  these  animals  for  trypanosomes.  Usually  at  the  time  the 
guinea-pigs  die  we  find  numerous  trypanosomes. 

Other  tests  are  (i)  Trypanolysis,  when  unheated  suspected  serum  and  trypano- 
somes are  incubated  together  for  one  hour.  Normal  serum  may  occasionally  cause 
disintegration  and  treated  cases  give  it  in  only  about  45%  of  cases.  Unfavorable 
untreated  cases  give  it  in  about  80%  of  cases. 

(2)  The  so-called  auto-agglutination  test  is  not  of  much  value.  In  this  the  red 
cells  of  the  blood  of  a  trypanosomiasis  case  come  together  in  clumps  when  one 
makes  a  wet  preparation.  It  is  not  a  rouleaux  formation.  (3)  The  attachment  test 
is  made  by  making  a  mixture  of  inactivated  serum,  leukocytes  and  trypanosomes 
and  allowing  them  to  be  in  contact  for  twenty  minutes.  A  positive  test  shows 
attachment  of  the  trypanosomes  to  the  leukocytes. 

Of  the  more  important  trypanosome  diseases  of  animals  may  be 
mentioned: 

1.  Nagana.    Pathogenic  for  domesticated  animals  in  South  Africa.     T.  brucei. 

2.  Surra.    Pathogenic  for  horses  in  India  and  Philippines.     T.  evansi. 

3.  Dourine.    Transmitted  by  coitus  in  horses.     T.  equiperdum. 

4.  Mai  de  caderas.    Affects  horses  in  South  America.     T.  equinum. 

A  harmless  infection,  especially  in  sewer  rats,  is  due  to  T.  levrisi.  Transmission  of 
this  rat  trypanosomiasis  can  apparently  be  brought  about  through  the  agency  of 
both  fleas  and  lice.  In  the  flea  there  is  apparently  a  developmental  cycle  of  a  dura- 
tion of  one  week. 

There  are  many  trypanosomes  in  birds,  fish,  frogs,  etc. 

Schizotrypanum  cruzi  (Trypanosoma  cruzi). — In  1909,  Chagas  re- 
ported the  finding  of  a  flagellate  in  the  intestines  of  Conorhinus  megistus 
or,  more  properly,  Lamus  megistus.  He  was  also  able  to  transmit  the 
flagellate  to  laboratory  animals  and  could  culture  it  on  blood  agar. 

In  investigating  the  matter  of  the  importance  of  this  flagellate, 
Schizotrypanum  cruzi,  in  Minas  Geraes,  Brazil,  where  the  bug  was  pres- 
ent in  great  numbers  in  the  cracks  of  the  houses  of  the  poor,  he  asso- 
ciated this  flagellate  infection,  which  he  at  first  considered  trypano- 
somal,  with  a  disease  of  the  children  of  that  section. 

The  bug  is  a  vicious  feeder  and,  from  its  biting  chiefly  about  the  face,  has  been 
called  barbiero  or  barber  by  the  natives.  Both  the  male  and  female  of  Lamus  bite 
and  can  transmit  the  disease  and  although  the  parasite  is  not  transmitted  hereditarily 
the  nymph  is  capable  of  sucking  blood  and  becoming  infected. 

It  requires  several  months  for  the  insect  to  go  through  the  egg,  larval  and  pupal 
stage  to  maturity.  Some  consider  this  bug  to  belong  to  the  genus  Triatoma.  The 
insects  may  live  for  more  than  a  year  and  tend  to  remain  in  the  same  house  where 


r  or 


BRAZILIAN  TRYPANOSOMIASIS  267 

they  may  have  become  infected  but  leave  such  house  if  it  be  abandoned  by  man 
Brumpt  thinks  that  the  bedbug  may  also  transmit  the  disease. 

S.  Cruzi  is  found  in  the  blood  of  children  during  the  acute  febrile 
stage  but  at  other  times  in  children,  and  as  a  rule  in  adults,  it  is  rarely 
present  in  the  peripheral  blood.     The  early  blood  forms  are  narrow 
and  very  motile.     They  increase  in  size  and  slacken  in  motility  when 
they  become  about  20  microns  long.     5.  cruzi  is  characterized  by  a 
very    large    blepharoplast.      Dividing 
forms  are  never  seen  in  the  blood.     The  \ 
common  site  of  multiplication  is  in  the    )     d£2\     /- 
cells  of  the  voluntary  muscles  and  heart   \zJr         ^^^        CV     (T^* 
and  also  in  the  cells  of  the  central  ner-  &f~\  \£g}) 

vous  system,  adrenals,  thyroid  and  bone 

marrow.     In  these  tissues  the  flagellate 

i    i  e  11  FIG.  62. — Schizotrypanum  cruzi 

takes  on  a  rounded  form  and  undergoes    in  blood  of  child  with  acute  type  of 

binary     division.      Continued     division     Brazilian  trypanosomiasis.     (Mac- 

.-,       •    p     ,    j        n    .    ,  Nealfrom  Doflein  after  Chaeas.) 

converts  the  infected  cell  into  a  cyst. 

It  is  this  process  going  on  in  various  important  structures  that  ac- 
counts for  the  extreme  variation  in  symptomatology  and  pathology. 

Chagas  thinks  that  the  gametes  for  the  cycle  in  Lamus  arise  from  parasites  develop- 
ing in  the  lungs  of  the  vertebrate  host.  Flagellated  parasites  enter  the  lungs,  lose  the 
flagellum  and  become  oval  in  shape,  later  on  dividing  into  8  parts.  These  assume 
an  elongated  form  and  enter  the  red  cells  of  the  host.  The  forms  taken  up  by 
Lamus  multiply  in  the  intestine  and  then  pass  to  the  salivary  glands  after  about  eight 
days.  The  bug  is  then  infectious  when  it  bites.  Brumpt  notes  that  infection  may 
occur  from  inoculation  of  the  faeces  passed  by  the  bug,  especially  through  the 
conjunctiva. 

Trypanoplasma 

The  genus  Trypanoplasma  has  a  rather  large  blepharoplast,  from  which  arise  two 
flagella.  One  extends  forward  as  a  free  anterior  flagellum,  while  the  other  projects 
posteriorly,  running  along  the  border  of  the  undulating  membrane.  This  genus  is 
not  known  for  man. 

Leishmania 

The  parasites  which  cause  a  general  infection  in  kala-azar  and 
leishmania  infantile  splenic  anaemia  but  a  local  one  in  oriental  sore 
are  usually  separated  as  distinct  species,  Leishmania  donovani  for 
kala-azar,  L.  infantum  for  infantile  splenic  anaemia  and  L.  tropica 
for  oriental  sore. 


268  THE   PROTOZOA 


. 

>  x 
^rt_ 


These  parasites  are  grouped  with  the  haemoflagellates  and  occur  in  their  verte- 
brate hosts  exclusively  as  small,  oval,  cockle-shell-shaped  bodies,  measuring  2.5 
3.0  microns.  The  protoplasm  stains  a  faint  blue  and  contains  a  rather  large  tropho- 
nucleus  which  is  peripherally  placed  and  gives  the  appearance  of  the  hinge  of  the 
cockle  shell.  Besides  this  macronucleus  we  have  a  second  chromatin  staining  body 
which  is  often  rod-shaped  and  set  at  a  tangent  to  the  larger  nuclear  structure.  It  is 
called  the  blepharoplast  or  micronucleus  and  stains  a  more  intense  reddish  than  the 
rather  fainter  stained  pinkish  macronucleus.  One  or  more  vacuoles  are  common  in 
the  cytoplasm. 

Some  consider  these  nonflagellated  bodies,  which  are  usually  found  packed  in 
endothelial  cells  of  spleen,  liver,  lymphatic  glands  and  bone  marrow,  as  resting  stages, 
the  flagellate  existence  occurring  in  some  other  host  than  its  vertebrate  one.  Patton 
has  carried  on  an  immense  amount  of  experimental  work  with  the  bedbug  and  has 
noted  the  development  of  flagellate  forms  from  the  fifth  to  the  eighth  days  in  bugs 
which  fed  on  kala-azar  patients  showing  leishman  bodies  in  their  peripheral  circula- 
tion. If  the  bugs  are  allowed  a  second  feeding  after  the  infecting  blood  meal  the 
flagellates  disappear  within  twelve  hours,  so  that  for  full  development  in  the  bedbug  a 
single  feeding  is  requisite.  He  states  that  the  flagellate  forms  change  to  post-flagel- 
lates ones  by  the  twelfth  day.  At  the  same  time,  although  much  evidence  exists  in 
favor  of  the  bedbug  as  host  for  the  flagellate  forms,  it  has  not  been  shown  experi- 
mentally that  the  bedbug  is  definitely  connected  with  the  transmission  of  the  disease. 

Donovan  is  disposed  to  incriminate  Conorrhinus  rubrifasciatus  as  the  transmitting 
agent  and  furthermore  he  feels  that  there  has  not  been  sufficient  investigation  of  mos- 
quitoes along  this  line. 

In  the  regions  where  leishmaniasis  of  infants  occurs  there  is  also 
found  a  similar  disease  of  dogs  and  Basil e  has  claimed  that  the  disease 
is  transmitted  from  dog  to  dog  by  the  dog  flea. 

As  the  dog  has  been  regarded  by  some  as  the  reservoir  of  the  virus  so  naturally  the 
transmission  of  the  disease  from  dog  to  child  through  the  flea  has  been  considered. 
Wenyon,  however,  tried  to  infect  two  young  dogs  with  great  numbers  of  fleas  which 
had  previously  fed  on  dogs  infected  with  canine  leishmaniasis  and  at  autopsy,  five  or 
six  weeks  later,  was  unable  to  find  parasites  in  smears  from  spleen,  liver  or  bone 
marrow  and  did  not  succeed  in  obtaining  cultures  from  this  material  inoculated  into 
tubes  of  N.  N.  N.  medium. 

As  regards  oriental  sore  Wenyon  has  found  that  bedbugs  and  Stegomyia  will  feed 
from  the  sores  and  take  up  parasites  which  develop  into  flagellate  forms  in  the  gut  of 
the  insects. 

Proof  of  transmission  by  these  agents,  however  is  lacking  and  others  are  inclined 
to  suspect  the  house  fly  or  some  species  of  moth  midge. 

In  Brazil  there  exists  some  evidence  that  the  cutaneous  leishmaniasis  found  there 
may  be  transmitted  by  species  of  the  tabanid  family. 

It  must  be  understood  that  there  is  always  a  suspicion  that  the 
flagellate  forms  noted  in  arthropod  experiments  may  be  those  of  non- 
pathogenic  herpetomonad  or  crithidial  species  as  such  forms  are 


LEISHMANIASIS  269 

common  in  arthropods  and  are  difficult  to  distinguish  from  the  flagellate 
stage  of  leishman  bodies. 

The  genera  Herpelomonas  (Leptomonas)  and  Crlthidia  are  frequently  found  in  the 
alimentary  tract  of  insects  and  have  caused  confusion  in  the  search  for  developmental 
forms  of  various  pathogenic  flagellates  in  transmitting  insects.  In  Herpetomonas,  of 
which  the  type  species  is  H.  muscoe  domestic^,  the  body  is  spindle-shaped  with  a 
rather  blunt  flagellar  end  and  an  attenuated  anterior  end.  In  Crithidia  both  extremi- 
ties are  pointed  and  the  blepharoplast  is  situated  toward  the  center  quite  near  the 
trophonucleus.  In  Herpetomonas  the  blepharoplast  is  near  the  rather  blunt 
flagellar  extremity  at  some  distance  from  the  nucleus. 

There  is  no  undulating  membrane  in  Herpetomonas  and  only  a  slightly  developed 
one  in  Crithidia  while  Trypanosoma  has  a  fully  developed  one.  In  Herpetomonas 
the  blepharoplast  is  near  the  flagellated  end  and  at  a  distance  from  the  trophonucleus. 
In  Crithidia  the  blepharoplast  is  more  posterior  and  near  the  macronucleus  but  still 
anterior  to  it  while  in  Trypanosoma  the  blepharoplast  has  moved  so  far  posteriorly 
as  to  pass  the  nucleus  and  be  located  at  the  posterior  extremity.  The  flagellated 
Leishmania  is  morphologically  herpetomonad. 

Very  definite  is  our  knowledge  of  the  cultural  forms  of  Leishmania.  Rogers  first 
cultured  material  from  splenic  juice  of  kala-azar  patients  in  10%  sodium  citrate  solu- 
tion at  a  temperature  of  1 7°  to  24°C.  The  medium  was  slightly  acidulated  with  citric 
acid.  There  was  no  satisfactory  development  at  blood  temperature.  In  forty-eight 
hours  the  oval  parasites  have  developed  into  herpetomonad  flagellates,  from  20  to 
22  microns  long  by  3  V£  microns  broad,  with  a  2o-micron  flagellum  which  takes  origin 
from  the  blunt  anterior  end  of  the  body  near  the  blepharoplast.  The  peripheral 
blepharoplast  and  centrally  placed  macronucleus  are  at  a  distance  from  one  another 
as  opposed  to  the  approximation  of  the  crithidial  blepharoplast  to  the  centrally 
placed  nucleus  in  a  body  with  pointed  anterior  end. 

Formerly  it  was  thought  that  there  were  differences  in  the  three 
species  of  Leishmania  from  the  standpoint  of  growth  on  various  culture 
media,  L.  donovani  not  growing  on  N.  N.  N.  medium  while  L.  infantum 
grew  well  on  N.  N.  N.  medium  but  not  in  citrated  blood.  It  is  now 
known  that  both  species  will  grow  on  these  two  media. 

It  is  absolutely  essential  in  culturing  L.  donovani  or  L.  infanttim  that  the  blood 
agar  or  citrated  blood  be  sterile,  as  any  bacterial  contamination  prevents  growth. 
With  the  parasite  L.  tropica,  however,  bacterial  contamination  does  not  inhibit 
development  and  statements  have  even  been  made  that  growth  is  favored  by  a 
staphylococcal  symbiosis.  L.  tropica,  it  would  seem,  will  develop  into  flagellated 
forms  in  cultures  at  28°C.  while  it  will  be  remembered  that  Rogers  in  his  original 
experiments  failed  to  obtain  other  than  commencing  signs  of  division  at  27°C.,  22°C. 
being  the  temperature  necessary  for  the  development  of  flagellate  forms. 

L.  tropica  from  South  American  cutaneous  leishmaniases  seems  to  grow  more  luxur- 
iantly on  N.  N.  N.  medium  than  does  that  of  oriental  sore  of  Asia  and  Africa. 

While  differences  in  development  on  different  culture  media  may 
obtain  not  only  with  different  species  but  with  different  strains  of  the 


270  THE    PROTOZOA 

same  species,  it  would  appear  that  such  variations  cannot  be  utilized 
as  a  means  of  separating  the  three  species. 

With  animal  inoculations  we  formerly  thought  that  the  parasite  of  kala-azar 
could  be  differentiated  from  that  of  infantile  leishmaniasis  by  the  fact  that  dogs 
could  not  be  infected  with  L.  donovani,  while  they  were  susceptible  to  infections 
with  L.  infantum.  Recently  Donovan  and  Patton  have  successfully  inoculated  dogs 
with  kala-azar  splenic  material.  Patton  found  the  parasites  in  the  liver,  spleen  and 
lymphatic  glands  as  well  as  bone  marrow  of  the  inoculated  dogs.  Consequently  we 
cannot  separate  the  two  visceral  leishmaniases  from  a  standpoint  of  susceptibility 
of  the  dog.  Monkeys  are  susceptible  to  both  diseases. 

As  regards  separating  oriental  sore  from  the  visceral  leishmaniases  Gonder  has 
shown  that  white  mice  may  be  infected  with  both  kala-azar  and  oriental  sore,  there 
being  produced  in  each  case  a  general  infection  with  the  presence  of  parasites  in 
spleen  and  liver.  A  point  of  difference,  however,  is  that  the  oriental  sore  mice 
develop  lesions  on  feet,  tail  and  head  which  was  not  observed  with  the  kala-azar 
mice.  There  are  some  reasons  for  thinking  that  in  human  cutaneous  leishmaniasis 
a  generalized  infection  may  precede  the  local  manifestations. 

A  very  interesting  point  is  that  the  dogs  in  India  never  show  a 
natural  infection  with  L.  donovani  while  in  the  regions  where  L.  in- 
fantum is  responsible  for  human  infections  the  natural  infection  of 
dogs  is  not  uncommon,  indeed  many  think  the  dog  the  reservoir  of 
virus  for  both  L.  infantum  and  L.  tropica.  It  has  been  suggested  that 
the  dogs  of  India,  where  kala-azar  prevails,  may  be  immune. 

As  regards  morphology  it  is  usually  stated  that  the  parasites  of  the  three  species  of 
Leishmania  are  practically  identical.  In  cultures  it  has  been  noted  that  the  flagella 
of  L.  tropica  are  longer  and  more  twisted  than  those  of  L.  infantum.  Again  it  has 
been  observed  that  the  parasites  of  the  Oriental  and  South  American  skin  lesions  may 
at  times  show  a  flattened  or  band-like  trophonucleus  instead  of  the  constant  round 
or  oval  one  of  the  visceral  leishmaniases. 

Escomel  has  reported  the  finding  of  flagellated  Leishmania  in  the  South  American 
sores. 

Laboratory  Diagnosis. — The  leukemias  can  be  easily  differentiated 
by  the  blood  picture,  an  important  matter  because  the  spleen  of 
splenomyelogenous  leukemia  is  very  friable  and  the  danger  from  splenic 
puncture  is  far  greater  in  this  condition  than  in  kala-azar.  Banti's 
disease  with  its  leucopenia  shows  a  rather  similar  blood  picture  and 
can  only  be  surely  differentiated  by  the  finding  of  leishman  bodies  in 
kala-azar. 

Malta  fever,  typhoid  and  the  paratyphoids  are  best  differentiated  by  blood  cul- 
tures or  agglutination  tests. 

Until  recently  it  was  recommended  that  for  diagnosis  our  best  procedure  was 


INTESTINAL   FLAGELLATES  271 

to  make  a  splenic  puncture.  Manson  and  others  have  pointed  out  the  dangers 
from  splenic  puncture  in  kala-azar  and  have  rather  preferred  puncture  of  the  liver, 
although  recognizing  that  the  chances  of  obtaining  parasites  from  a  liver  puncture, 
are  less  than  from  a  splenic  one. 

Statistics  have  been  given  where  a  mortality  approximating  i%  has  followed 
spleen  puncture.  Bousfield,  however,  using  an  all  glass  syringe  with  a  i^-inch 
needle  did  not  have  a  fatality  in  1 20  spleen  punctures. 

For  diagnosis  the  spleen  or  liver  juice,  rather  than  pure  blood,  is 
smeared  on  a  slide  and  stained  by  some  Romanowsky  method,  pref- 
erably that  of  Giemsa. 

Cultures  on  N.  N.  N.  medium  can  also  be  made. 

One  should  always  first  examine  a  smear  of  the  peripheral  blood  for  parasites  in 
polymorphonuclear  or  large  mononuclear  leukocytes.  The  Sudan  Commission 
found  leishman  bodies  in  the  peripheral  blood  of  13  out  of  15  cases  so  examined,  but 
rarely  did  they  find  more  than  one  parasite-containing  leukocyte  to  a  slide. 

Quite  recently  Wenyon  and  others  have  noted  the  desirability  of 
culturing  the  peripheral  blood  in  N.  N.  N.  medium.  Diagnosis  may 
be  made  in  this  way,  provided  one  wait  from  two  to  three  weeks 
before  reporting  negatively  as  to  the  presence  of  flagellated  Leishmania 
in  the  cultures.  As  before  stated,  strict  asepis  and  a  room  temperature 
are  requisite  for  flagellate  development. 

It  has  been  noted  that  artificial  pustulation  might  assist  in  diagnosis  by  giving 
a  multitude  of  polymorphonuclear  leukocytes  for  examination  for  phagocytized 
Leishmania. 

Cochran  has  recently  noted  the  advisability  of  excising  a  lymphatic  gland  and 
making  gland  smears  to  examine  for  Leishmania.  Others  have  reported  success 
with  gland  puncture  as  utilized  in  the  glands  of  trypanosomiasis. 

NOTE. — Darling  has  reported  from  Panama  a  protozoon  somewhat 
like  Leishmania  in  which  the  cells  .of  lungs,  liver,  spleen,  and  lym- 
phatic glands  contained  numerous  parasites  about  3  to  4^  in  diameter, 
slightly  oval  in  outline,  and  containing  a  large  and  small  chromatin 
staining  mass.  He  has  given  it  the  name  Histoplasma  capsulata. 

INTESTINAL  FLAGELLATES 

These  parasites  of  the  intestinal  tract  are  separated  according  to 
the  number  of  their  flagella. 

These  flagella  can  easily  be  counted  in  a  preparation  mounted  in  Gram's  iodine 
solution.  For  this  purpose  I  take  a  clean  slide  and  make  a  vaseline  line  across  it 
about  i  inch  from  the  end.  A  drop  of  the  iodine  solution  is  placed  on  the  slide  about 


272  THE   PROTOZOA 

%  inch  from  the  vaselined  line  and  a  suitable  portion  of  the  faeces  to  be  examii 
is  emulsified  in  it.  The  edge  of  a  square  cover-glass  is  then  applied  to  the  vaselined 
line  and  allowed  to  drop  on  the  preparation.  By  pressure  suitable  thicknesses  of 
fluid  can  be  examined.  There  is  an  absence  of  current  motion.  Better,  when  access- 
ible, is  it  to  use  the  dark  field  illuminator  as  in  this  way  the  flagella  are  distinctly 
brought  out.  The  india  ink  method  is  also  applicable.  Staining  of  smears  by 
Giemsa's  method,  following  fixation  in  methyl  alcohol  or  5%  formalin  solution  is 
more  satisfactory  for  flagellates  than  for  amoebae,  which,  as  before  stated,  should 
be  fixed  in  moist  smears  and  stained  by  haematoxylin.  This  method  of  mounting 
in  iodine  solution,  however,  is  the  one  I  always  use  for  encysted  amoebae. 

The  intestinal  flagellates  are  classified  according  to  number  of 
flagella,  absence  or  presence  of  an  undulating  membrane  and  of  a 
blepharoplast.  Three  of  these  flagellates  are  but  rarely  found  in  the 
stools  and  seem  to  be  of  little  importance.  They  are  (i)  Cercomonas, 
which  has  a  single  nucleus  with  one  free  flagellum  and  a  second  one 
which  turns  backward  to  be  attached  to  the  body  and  then  projects 
posteriorly  as  a  second  free  flagellum,  (2)  Bodo,  which  has  a  single  nuc- 
leus, but  two  anteriorly  projecting  flagella  and  (3)  Prowazekia  which 
has,  besides  the  nucleus,  a  blepharoplast  from  which  arise  two  flagella. 
These  flagellates  can  be  cultivated  on  media  used  for  the  cultural 
amoebae  and  it  is  thought  by  some  that  they  at  times  show  amoebae- 
like  stages. 

There  is  an  organism,  supposed  to  belong  to  the  moulds,  which  may  be  mistaken 
for  an  encysted  flagellate.  It  is  called  Blastocystis  hominis  and  has  a  large  central 
vacuole  with  a  refractile  narrow  rim  which  contains  one  or  more  nuclei.  When 
stained  by  Giemsa's  stain  the  central  part  is  very  faintly  stained  while  the  rim  is 
deep  blue. 

Trichomonas  intestinalis. — This  is  a  very  common  parasite  in  diarrhceal  stools 
but  as  to  its  pathogenicity  there  is  much  doubt.  It  is  pear-shaped  and  about  9  by 
14  microns.  There  are  three  flagella  projecting  anteriorly  with  a  fourth  one  bordering 
an  undulating  membrane  and  projecting  posteriorly.  It  has  a  cytostome  near  the 
nucleus.  There  is  also  a  T.  vaginalis  which  is  found  in  vaginal  secretion  of  acid 
reaction,  disappearing  when  the  reaction  becomes  alkaline  as  at  the  time  of 
menstruation.  It  is  somewhat  larger  than  the  intestinal  form  and  is  not  infre- 
quently found  in  urine. 

Tetramitus  mesnili. — This  flagellate  differs  from  the  preceding  one  in  not  having 
an  undulating  membrane  or  fourth  flagellum.  The  three  anteriorly  projecting 
flagella  are  long  and  slender.  There  is  a  very  prominent  long  slit-like  cytostome 
within  which  is  a  flagellum.  The  nonflagellate  end  is  very  much  attenuated. 

This  parasite  has  been  reported  not  infrequently  as  a  cause  of  diarrhceal  condi- 
tions since  its  first  reporting  by  Wenyon  in  1910. 

All  the  above-mentioned  flagellates  are  found  in  the  large  intestines  especially  in 
the  region  of  the  caecum. 


CTLIATES 


Lamblia 


273 


Lamblia  intestinalis.— These  parasites  are  about  10  X  ISM  and  have  a  pear-shaped 
body  with  a  depression  at  the  blunt  anterior  end.  This  depression  enables  the 
flagellate  to  attach  itself  to  the  summit  of  an  epithelial  cell.  Around  the  depression 
are  three  pairs  of  flagella  which  are  constantly  in  motion.  Another  pair  of  flagella 
project  from  either  side  of  the  blunt  little  tail-like  projection.  When  stained,  the 
parasites  have  a  pyriform  shape  with  two  chromatin  staining  areas  on  either  side 
of  the  anterior  end. 

In  motion  they  have  a  slow  tumbling  sort  of  progression.  These  parasites  live 
in  the  upper  portion  of  the  small  intestines.  The  cysts  are  oval  and  show  the 
folded  flagellate  within.  There  is  an  appearance  of  two  curved  lines  and  two  dots. 
This  infection  is  frequently  associated  with  a  debilitating  diarrhoea.  Some  cases 
show  marked  nervous  symptoms.  In  examining  the  stools  of  384  cases,  who  had 
practically  all  been  in  Gallipoli  or  Egypt,  Woodcock  and  Penfold,  in  the  King  George 
Hospital,  found  98  infected  with  protozoa  as  follows:  Lamblia,  22;  Trichomonas,  14; 
Tetramitus  (Macrostoma),  n;  E.  coli,  57;  E.  histolytica,  8;  Isospora,  10. 

INFUSORIA  (CILIATA) 
The  Infusoria  are  the  most  highly  developed  of  the  Protozoa. 

The  bodies  of  Infusoria  are  oval  and  may  be  free  or  attached  to  a  stalk-like  con- 
tractile pedicle,  as  with  Vorticella,  or  they  may  be  sessile.  The  cilia,  which  are 
characteristic,  may  be  markedly  developed  around  the  cytostome  (mouth)  and  serve 
the  purpose  of  directing  food  into  the  interior,  while  others  act  as  locomotor  organs. 
The  body  is  enveloped  by  a  cuticle  which  may  only  have  one  opening  or  slit,  to  serve 
as  mouth;  or  it  may  have  a  second  one,  a  cytopyge  or  anus.  Usually  the  faecal 
matter  is  ejected  through  a  pore  which  may  be  visible  only  when  in  use.  They  usu- 
ally have  a  large  nucleus  and  a  small  one.  Infusoria  tend  to  encyst  when  conditions 
are  unfavorable  (as  when  water  dries  up  in  a  pond).  When  the  cilia  are  evenly 
distributed  over  the  entire  body  of  the  cilia tes  we  have  the  order  Holotricha;  when 
ciliated  all  over,  but  with  more  prominent  cilia  surrounding  the  peristome,  we  call 
the  order  Heterotricha.  It  is  to  this  order  that  the  Infusoria  of  man  belong. 

Balantidium  coli. — This  is  the  only  cilia te  of  importance  in  man. 
It  is  a  common  parasite  of  hogs.  It  is  from  60  to  ioo/*  long  by  50  to 
yo/*  broad,  and  has  a  peristome  at  its  anterior  end  which  becomes  narrow 
as  it  passes  backward.  It  has  an  anus.  The  ectosarc  and  the  endosarc 
are  distinctly  marked.  The  cuticle  is  longitudinally  striated. 

These  parasites  cause  an  affection  similar  to  dysentery  and  may  bring  about  a 
fatal  termination.  It  is  almost  impossible  to  escape  noticing  the  actively  moving 
bodies  if  a  fgecal  examination  is  made.  When  encysted  they  are  round. 

Another  ciliate,  the  B.  minimum,  25  X  i5M,  has  also  been  reported  for  man. 

Nyctotherus  faba  has  a  kidney-shaped  body  and  is  about  25  by  15/1.  It  has  a 
large  contractile  vacuole  at  the  posterior  end.  It  has  a  large  nucleus  in  the  center 
18 


DESCRIPTION  OF  PLATE  I 

(Kolle  and  Wassermann) 
Malarial  Parasites 

1.  Two  tertian  parasites  about  thirty-six  hours  old,  attacked  blood-corpuscles 
swollen. 

2.  Tertian  parasite  about  thirty-six  hours  old;  stained  by  Romanowsky's  method. 
The  black  granule  in  the  parasite  is  not  pigment  but  chromatin.     Next  to  it  and  to 
the  left  is  a  large  lymphocyte,  and  under  it  the  black  spot  is  a  blood  plate. 

3.  Tertian  parasite,  division  form  nearby  is  a  polynuclear  leukocyte. 

4.  Quartan  parasite,  ribbon  form. 

5.  Quartan  parasite,  undergoing  division. 

6.  Tropical  fever  parasite.     (^Estivo-autumnal.)     In  one  blood-corpuscle  may 
be  seen  a  smaller,  medium,  and  large  tropical  fever-ring  parasite. 

7.  Tropical  fever  parasite.     Gametes  half-moon  spherical  form.     Smear  from 
bone  marrow. 

8.  Tropical  fever  parasite  which  is  preparing  for  division  heaped  up  in  the  blood 
capillaries  of  the  brain. 

Asexual  Forms 

9.  Smaller  tertian  ring  about  twelve  hours  old. 

10.  Tertian  parasite  about  thirty-six  hours  old,  so-called  amoeboid  form. 

11.  Tertian  parasite  still  showing  ring  form  forty-two  hours  old. 

12.  Tertian  parasite,  two  hours  before  febrile  attack.     The  pigment  is  beginning 
to  arrange  itself  in  streaks  or  lines. 

13.  Tertian  parasite  further  advanced  in  division.     Pigment  collected  in  large 
quantities. 

14.  Further  advanced  in  the  division.     (Tertian  parasite.) 


274 


PLATE  I. 


DESCRIPTION  OF  PLATE  H 

(Kolle  and  Wassermann) 
Malarial  Parasites 

15.  Complete  division  of  the  parasite.     Typical  mulberry  form. 

1 6.  To  the  left  is  the  completed  division  form,  an  almost  developed  gamete,  which 
is  to  be  recognized  by  its  dispersed  pigment. 

17.  A  tertian  ring  parasite,  small  size  broken  up. 

1 8.  Threefold  infection  with  tertian  parasite.    The  oval  black  granules  are  the 
chromatin  granules. 

19.  To  the  left,  tertian  parasite  with  large,  sharply  demarked,  and  deeply  colored 
chromatin  granules.     To  the  right,  tertian  parasite.     Both  thirty-six  hours  old. 
Both  probably  gametes. 

20.  Tertian  parasite  thirty-six  hours  old,  ring  form. 

21.  Tertian  parasite  with  beginning  chromatin  division,  with  eight  chromatin 
segments. 

22.  Tertian  parasite  chromatin  division  farther  advanced  with  twelve  chromatin 
granules,  in  part  triangular  in  form. 

23.  Completed  division  figure  of  a   tertian  parasite.     Twenty-two   chromatin 
granules. 

24.  The  young  tertian  parasites  separating  themselves  from  each  other.     The 
pigment  remains  behind  in  the  middle. 

25.  Quartan  ring  parasite,  which  is  hard  to  differentiate  from  large  tropical  ring 
or  small  tertian  ring. 

26.  Quartan  ring  lengthening  itself. 

27.  Small  quartan  ribbon  form. 

28.  The  quartan  ribbon  increases  in  width.     The  dark  places  consist  almost 
entirely  of  pigment. 


276 


PLATE  II. 


DESCRIPTION  OF  PLATE  HI 

(Kolle  and  Wassermann) 
Malarial  Parasite 

29>  3°>  31-     The  quartan  ribbon  increases  in  width.     The  dark  places  consist 
almost  entirely  of  pigment. 

32.  Beginning  division  of  the  quartan  parasite  and  the  black  spot  in  the  middle 
is  the  collected  pigment. 

33.  Quartan  ring. 

34.  Double  infection  with  quartan  parasites. 

35.  Wide  quartan  band.     The  fine  black  stippling  in  the  upper  half  of  the  parasite 
is  pigment. 

36.  Beginning  division  of  the  quartan  parasite.     The  chromatin  (black  fleck) 
is  split  into  4  parts. 

37.  Division  advanced,  quartan  parasites. 

38.  Typical  division  figure  of  the  quartan  parasite. 

39.  Finished  division  of  the  quartan  parasite.     Ten  young  parasites,  pigment  in 
the  middle. 

40.  Young  parasites  separated  from  one  another. 

41.  Small  and  medium  tropical  ring,  the  latter  in  a  transition  stage  to  a  large 
tropical  ring. 

42.  Small,  medium  and  large  tropical  ring,  together  in  one  corpuscle. 


278 


PLATE  III. 


DESCRIPTION  OF  PLATE  IV. 

(Kolle  and  Wassermann.) 
Malarial  Parasite. 

43.  To  the  left  a  young  tropical  parasite.     To  the  right  a  medium  and  large 
tropical  parasite. 

44.  An  almost  fully  developed  tropical  parasite.    The  black  granules  are  pig- 
ment heaps. 

45.  Young  parasites  separated  from  one  another.     Broken  up  division  forms 
twenty-one  new  parasites. 

46.  To  the  left  a  red  blood-corpuscle  with  basophilic,  karyochromatophilic  gran- 
ules.    Prototype  of  malarial  parasite.     On  the  right  a  red  blood-corpuscle  with  re- 
mains of  nucleus. 

Sexual  Forms  or  Gametes. 

47.  An  earlier  quartan  gamete  (macrogamete  in  sphere  form),  female. 

48.  An  earlier  quartan  gamete  (microgametocyte),  male. 

49.  Tertian  gamete,  male  form  (microgametocyte). 

50.  Tertian  gamete,  female  (macrogamete). 

51.  Tertian  gamete  (microgametocyte)  still  within  a  red  blood-corpuscle. 

52.  Macrogamete  tertian  within  a  red  blood-corpuscle. 

53.  Tropical    fever.     (^Estivo-autumnal)    gamete,    half-moon    (crescent)    still 
lying  in  a  red  blood-corpuscle.     In  the  middle  is  the  pigment.     The  concave  side 
of  the  crescent  is  spanned  by  the  border  of  the  red  blood-corpuscle. 

54.  Gamete,  tropical  fever  parasite. 

55.  Gamete  of  tropical  fever  parasite  heavily  pigmented. 

56.  Gamete  of  the  tropical  fever  parasite  (flagellated  form),  microgametocyte 
sending  out  microgametes  (flagella  or  spermatozoa). 


280 


PLATE  IV. 


282  THE   PROTOZOA 

with  a  small  fusiform  micronucleus  lying  close  to  it.     It  has  only  been  reported 
once  for  man. 


SPOROZOA 


This  class  of  Protozoa  gets  its  name  from  the  method  of  reproduction 
— sporulation.  These  parasites  rarely  show  binary  fission.  While 
the  sporozoa  are  found  within  cells,  in  the  tissues  and  in  internal  cavi- 
ties, as  intestine  and  bile  ducts,  yet  it  is  as  inhabitants  of  the  blood  that 
they  have  their  greatest  importance  for  man — these  are  known  as 
Haemospbridia.  A  sporozoon  may  be  either  naked  or  amoeboid  or  be 
covered  with  a  distinct  cuticle. 

NOTE. — Sporozoa  are  divided  into  two  subclasses — the  Telosporidia  and  the  Neo- 
sporidia.  In  the  former  the  vegetative  activity  of  the  protozoon  goes  on  to  full 
growth  at  which  time  the  reproductive  activity  commences.  With  the  Neosporidia, 
however,  the  growth  and  reproduction  go  on  at  the  same  time. 

Among  the  Telosporidia  we  have  the  orders  Gregarinaria,  Coccidiaria,  and 
Haemosporidia. 

Gregarines  are  chiefly  parasites  of  arthropods  and  worms  and  are  not  known  for 
man  or  the  higher  vertebrates. 

The  subclass  Neosporidia  is  practically  of  no  importance  in  human  parasitology, 
only  the  order  Sarcosporidia  having  been  reported  for  man.  From  an  economic 
standpoint,  however,  the  order  Myxosporidia  is  of  great  importance — Nosema 
bombycis  being  the  cause  of  pebrine,  a  disease  destructive  to  the  silkworm. 

Coccidiaria 

The  parasites  of  the  order  Coccidiaria  are  almost  exclusively  found  in 
the  intestines  and  in  the  organs  connected  with  it.  In  the  vegetative 
stage  it  lives  within  an  epithelial  cell,  which  it  destroys.  Afterward 
it  falls  into  the  lumen  lined  by  this  epithelial  cell  and  sporulates,  either 
by  the  method  of  schizogony  or  sporogony. 

Owing  to  their  egg-like  shape,  coccidia  have  often  been  considered  as  the  ova  of 
intestinal  parasites,  and  vice  versa.  Upon  swallowing  an  oocyst  with  its  contained 
sporozoites  the  membrane  of  the  oocyst  is  digested  in  the  duodenum  and  the  sporo- 
zoites  liberated.  They  enter  epithelial  cells,  as  of  intestine,  and  reproduce  by 
schizogony.  After  a  varying  number  of  nonsexual  cycles  sporogony  commences, 
sporonts  being  produced  instead  of  schizonts.  The  female  sporont  is  fertilized  by 
the  microgamete  which  is  an  elongated  body  provided  with  two  flagella.  These 
microgametes  are  formed  from  the  male  sporont  and  when  thrown  off  from  the 
periphery  they  enter  (usually  a  single  one)  the  macrogamete.  After  fertilization  a 
resistant  membrane  is  formed  and  the  term  oocyst  is  used.  Within  the  oocyst 
are  found  smaller  cysts,  the  sporocysts,  in  which  the  sporozoites  are  formed. 

The  cycle  is  very  similar  to  that  of  malaria  except  that  no  arthropod  host  is 


THE   MALARIAL  PARASITE  283 

required  for  the  sexual  cycle.  The  spores  which  are  formed  in  schizogony  are  known 
as  merozoites. 

Merozoites  may  best  be  distinguished  from  sporozoites  by  the  presence  of  a 
nuclear  karyosome,  this  being  absent  in  sporozoites.  In  Eimeria  we  have  the 
oocyst  containing  four  sporocysts  with  two  sporozoites  in  each  sporocyst  while  in 
Isospora  we  have  an  oocyst  containing  two  sporocysts  with  four  sporozoites  in  each. 

Eimeria  stiedae. — This  sporozoon  is  usually  known  as  the  Coccidium  cuniculi 
or  C.  omforme.  It  is  most  frequently  found  in  the  epithelium  of  the  bile  ducts. 
It  has  very  rarely  been  reported  for  man.  In  these  cases  (about  five)  cysts  of  the 
liver  have  been  found  containing  coccidia.  The  parasite  is  about  40  X  20/z,  and  is 
oval  in  shape  with  a  double  outlined  integument.  The  sporozoites,  which  form 
inside,  are  falciform  in  shape.  These  escape  and  enter  fresh  epithelial  cells,  and 
thus  the  process  of  schizogony  goes  on.  The  parasites  of  the  liver  are  larger  than 
those  found  in  the  intestines,  these  latter  being  only  about  30  X  15/1-  In  the  fceces 
the  form  most  often  found  is  the  oocyst,  about  40  X  2o/*.  Infection  takes  place  by 
ingestion  of  the  oocyst. 

Wenyon  states  that  the  Eimeria  oocysts,  found  in  cases  from  Gallipoli,  are  round 
instead  of  the  usual  oval.  They  are  about  20  microns  in  diameter  and  contain  four 
sporocysts,  10X7  microns,  each  of  which  has  two  sporozoites  with  one  or  two  highly 
refractile  residual  bodies. 

Isospora  bigemina. — This  parasite,  formerly  called  the  Coccidium  bigeminum, 
lives  in  the  intestinal  villi  of  dogs  and  cats.  It  is  about  12X8/4  and  shows  a  highly 
refractile  envelope  (oocyst)  containing  two  biscuit-shaped  sporocysts  within  each  of 
which  are  four  sporozoites.  It  has  been  reported  for  man  three  times. 

Cases  from  the  Gallipoli  district  also  showed  oocysts  of  Isospora  with  two  sporocysts 
and  four  sporozoites  in  each. 

Haemosporidia 

Of  the  Sporozoa  found  in  the  blood  (Haemosporidia),  the  malarial 
parasites  are  the  only  ones  connected  with  disease  in  man. 

There  are  at  least  three  species  of  animal  parasites  which  produce  human  malaria, 
Plasmodium  vivax,  the  cause  of  benign  tertian,  P.  malaria  of  quartan  and  P.  falci- 
parum  of  sestivo-autumnal.  These  parasites  belong  to  the  haemamceba  type  of  the 
order  Heemosporidia,  of  the  class  Sporozoa  and  of  the  phylum  Protozoa. 

This  type  of  Haemosporidia  is  characterized  by  invasion  of  red 
cells,  amoeboid  movement,  pigment  production  and  the  extrusion  of 
flagellum-like  processes  from  the  male  sporont  after  the  blood  is  taken 
from  the  animal  and  allowed  to  cool. 

Other  Hajmospordia  which  are  very  important  in  diseases  of  domesticated  animals, 
but  not  for  man,  are  those  of  the  piroplasm  type. 

These  parasites  of  the  red  cells  do  not  produce  pigment  and  do  not  "exflagellate." 
It  is  to  parasites  of  this  type  that  some  authorities  have  ascribed  the  cause  of  black 
water  fever,  a  condition  undoubtedly  connected  with  malaria. 


284  THE   PROTOZOA 


It  has  been  thought  proper  by  some  to  consider  the  malarial  para- 
sites as  belonging  to  two  genera,  the  genus  Plasmodium,  characterized 
by  round  sexual  forms  and  including  P.  vivax  and  P.  malaria  and  the 
genus  Laverania,  characterized  by  crescent-shaped  sexual  forms  and 
including  but  one  species  L.  malaria,  that  of  aestivo-autumnal  malaria. 

In  addition  to  man,  infections  with  parasites  of  a  similar  nature  are  found  in  mon- 
keys (Plasmodium  kochi;  the  sexual  forms  alone  seem  to  be  present),  in  birds 
(Hcsmamaeba  relicta;  this  organism  is  usually  designated  Proteosoma).  An  infection 
of  crows  and  pigeons  of  like  nature  is  Halteridium.  Numerous  haemosporidia  have 
been  reported  for  bats,  various  other  mammals,  tortoises,  lizards,  etc. 

The  life  history  of  the  malarial  parasite  is  one  of  the  most  interesting  chapters  in 
medicine.  Laveran  discovered  the  parasite  in  1880.  In  1885,  Golgi  noted  that 
sporulation  occurred  simultaneously  at  time  of  malarial  paroxysm.  Koch,  Golgi, 
and  Celli  demonstrated  existence  of  different  species  for  different  types  of  fever. 
King  and  Laveran  (1884)  considered  possibility  of  mosquito  transmission.  Manson 
(1894)  formulated  hypothesis  that  gametes  were  destined  to  undergo  development  in 
the  mosquito  from  observing  that  flagellated  bodies  only  appeared  some  time  after 
the  blood  was  withdrawn. 

Ross  (1895)  demonstrated  that  flagellation  takes  place  in  the  stomach  of  the  mos- 
quito. McCallum  (1897)  saw  fertilization  of  macrogametes  by  microgametes  of 
Halteridium.  Opie  recognized  differences  in  sexual  characteristics. 

Ross  (1898)  demonstrated  life  cycle  of  bird  malaria  (Proteosoma},  showing  for- 
mation of  zygotes  and  presence  of  sporozoites  in  salivary  glands.  Grassi  and  Big- 
nami  proved  the  cycle  for  Anophelinas  for  human  malaria.  In  1900  (Sambon  and 
Low),  infected  mosquitoes  from  Italy  were  sent  to  London,  where,  by  biting,  they 
infected  two  persons. 

Life  History. — Malaria  can  be  transmitted  by  subcutaneous  or 
intravenous  injection  of  the  blood  of  a  patient  with  the  disease  into  a 
well  person,  the  same  type  being  reproduced. 

Such  a  method  of  transmission  is  only  of  scientific  interest  and  the 
regular  method  is  as  follows:  An  infected  anopheline  at  the  time  of 
feeding  on  the  human  blood  introduces  through  a  minute  channel  in  the 
hypopharynx  the  infecting  sporozoite  of  the  sexual  cycle. 

When  man  is  first  infected  by  sporozoites  we  have  starting  up  a  nonsexual  cycle, 
which  is  completed  in  from  forty-eight  to  seventy-two  hours,  according  to  the  species 
of  the  parasite.  The  falciform  sporozite  bores  into  a  red  cell,  assumes  a  round  shape 
and  continues  to  enlarge  (schizont).  Approaching  maturity,  it  shows  division  into  a 
varying  number  of  spore-like  bodies.  At  this  stage  the  parasite  is  termed  a  mero- 
cyte.  When  the  merocyte  ruptures,  these  spore-like  bodies  or  merozoites  enter  a 
fresh  cell  and  develop  as  before. 

Malarial  Toxin. — At  the  time  that  the  merocyte  ruptures  it  is 
supposed  that  a  toxin  is  given  off  which  causes  the  malarial  paroxysm. 


SEXUAL  CYCLE  OF  MALARIAL  PARASITE  285 

Rosenau,  by  injecting  intravenously  filtered  blood,  taken  from  a  patient  at  the 
time  of  sporulation  of  the  parasites  caused  a  malarial  paroxysm.  No  parasites 
developed  later.  Another  man  who  received  a  small  amount  of  unfiltered  blood 
showed  a  slight  paroxysm  and  four  days  later  showed  parasites  in  his  blood.  Hence 
the  parasite  will  not  pass  through  the  pores  of  a  Berkefeld  filter. 

The  cycle  goes  on  by  geometric  progression  from  the  first  introduc- 
tion of  the  sporozoite,  but  it  is  usually  about  two  weeks  before  a 
sufficient  number  of  merocytes  rupture  simultaneously  to  produce 
sufficient  toxin  for  symptoms  (period  of  incubation).  This  cycle  is 
termed  schizogony.  It  is  considered  that  there  must  be  several 
hundred  parasites  per  cubic  millimeter  sporulating  to  be  capable  of 
producing  symptoms.  ^ 

Gametes. — After  a  varying  tfme,  whether  by  reason  of  necessity  for  renewal  of 
vigor  of  the  parasite  by  a  respite  from  sporulation,  or  whether  from  a  standpoint  of 
survival  of  the  species,  sexual  forms  (gametes)  develop.  Some  think  that  sporozoites 
of  sexual  and  nonsexual  character  are  injected  at  the  same  time.  It  is  usually  con- 
sidered, however,  that  sexual  forms  develop  from  preexisting  nonsexual  parasites. 
The  developing  gametes  are  often  termed  sporonts.  Strictly,  the  sexual  parasites 
in  the  blood  should  be  called  gametocytes.  The  gametes  take  about  twice  as  long  to 
reach  maturity  as  schizonts.  The  life  of  a  crescent  has  been  estimated  as  about  ten 
days  and  that  of  the  gametes  of  benign  tertian  and  quartan  about  one-half  this  period. 

The  gametes  show  two  types:  the  one  which  contains  more  pigment,  has  less 
chromatin,  and  stains  more  deeply  blue  is  the  female — a  macrogametocyte;  the  other 
with  more  chromatin,  less  pigment,  and  staining  grayish  green  or  light  blue  is  the 
male — a  microgametocyte.  When  the  gametes  are  taken  into  the  stomach  of  the 
Anophelinse,  the  male  cell  throws  off  spermatozoa-like  projections,  which  have  an 
active  lashing  movement  and  break  off  from  the  now  useless  cell  carrier  and  are 
thereafter  termed  microgametes.  These  fertilize  the  macrogametes  and  this  body 
now  becomes  a  zygote.  (Following  nuclear  reduction  with  formation  of  polar  bodies 
the  macrogametocyte  becomes  a  macrogamete.)  This  process  of  exflagellation  can 
be  observed  in  a  wet  preparation  under  the  microscope.  There  is  first  seen  a  very 
active  movement  of  the  pigment  of  the  male  gamete  and  finally  long  delicate  bulbous 
tipped  flagellum-like  processes  are  thrown  off  (exflagellated)  and  push  aside  the  red 
cells  by  their  progressive  motion.  McCallum  saw  a  female  Halteridium  fertilized 
by  the  microgamete,  after  which  it  was  capable  of  a  worm-like  motion  (vermiculus  or 
ookinete). 

By  a  boring-like  movement  the  vermiculus  stage  of  the  zygote  goes  through  tt 
walls  of  the  mosquito's  stomach,  stopping  just  under  the  delicate  outer  layer  of  the 
stomach  or  mid-gut.  In  three  or  four  days  after  fertilization  the  zygote  becomes 
encapsulated  and  is  then  often  called  an  oocyst.  It  continues  to  enlarge  until  at 
about  the  end  of  one  week  it  has  grown  to  be  about  SOM  in  diameter  and  has  be- 
come packed  with  hundreds  of  delicate  falciform  bodies.  Some  only  contain 
a  few  hundred,  others  several  thousand. 

Zygotes.-- In  some  of  his  observations  Darling  has  noted  that  the  zygote  of  benign 
tertian  malaria  grows  larger  and  more  rapidly  than  that  of  sestivo-autumnal  and  that 


286 


THE    PROTOZOA 


mn  o  1 


the  pigment  is  clumped  rather  than  in  belts  or  lines  as  with  aestivo-autumnal. 
Darling  has  also  noted  that  mosquitoes  do  not  tend  to  become  infected  unless  the 
gamete  carrying  man  has  more  than  1 2  gametes  to  the  cubic  millimeter. 

The  capsule  of  the  mature  zygote  ruptures  about  the  tenth  day  and  the  sporo- 
zoites  are  thrown  off  into  the  body  cavity.  They  make  their  way  to  the  salivary 
glands  and  thence,  by  way  of  the  veneno-salivary  duct,  in  the  hypopharynx,  they  are 
introduced  into  the  circulation  of  the  person  bitten  by  the  mosquito,  and  start  a 
nonsexual  cycle.  As  the  sexual  life  takes  place  in  the  mosquito,  this  insect  in  the 
definitive  host  and  man  is  only  the  intermediate  host.  The  cycle  in  the  mosquito 
takes  about  ten  to  twelve  days. 


e 

forms  in  Man 
~  Gametes  — 


>/oheline  mosquito  (definitive 
neriod  tcLsts  Q-/odays 
t  25' 'to 30"  C. 


-sexua/  cyc/e     0      f 
\jn  man  (inter-  \  Q  ' 
J^         mediary  Aostjo0  o£ 

^— ,. 

®-cg 


FIG.  63. — Sexual  (sporogony  in  mosquito)  and  non-sexual  (schizogony  in  man) 
cycle  of  the  malarial  parasite.  The  sporogony  diagram  to  the  left  shows  in  lower 
portion  the  fertilization  of  the  female  gamete  by  the  microgamete.  The  vermiculus 
stage  of  the  zygote  is  shown  boring  into  the  walls  of  the  mosquito's  stomach  to  later 
become  the  more  mature  zygote  packed  with  sporozoites  as  shown  in  the  upper  dia- 
gram of  the  developmental  processes  in  the  mosquito's  stomach. 

Efficient  Mosquito  Hosts. — It  must  be  remembered  that  only  certain 
genera  and  species  of  Anophelinae  are  known  malaria  transmitters; 
thus  Stephens  and  Christophers,  in  dissecting  496  mosquitoes  of  the 
species  M.  rossi,  did  not  find  a  single  gland  infected  with  sporozoites. 
With  M.  culicifacies,  however,  12  in  259  showed  infection.  A 
mosquito  which  is  capable  of  carrying  out  the  complete  sporogonous 
cycle  is  an  efficient  host  and  in  the  case  of  malaria  the  mosquito  is  the 
definitive  host  (sexual  life  of  parasite). 

Malarial  Index. — Mosquito  dissection  is  one  method  of  determining 
the  endemicity  of  malaria  or  the  malarial  index.  There  are  two  other 
methods:  i.  by  noting  the  prevalence  of  enlarged  spleens,  and  2.  by 


MALARIAL  INDEX  287 

determining  the  number  of  inhabitants  showing  malarial  parasites  in 
the  blood.  This  index  is  best  determined  from  children  between  two 
and  ten  years  of  age,  as  children  under  two  show  too  high  a  proportion 
of  parasites  in  the  peripheral  blood  while  those  over  ten  years  of  age 
show  too  great  an  incidence  of  enlarged  spleens. 

As  before  stated  there  are  three  species  of  malarial  parasites:  i.  Plasmodium 
vivax,  that  of  benign  tertian — cycle,  forty-eight  hours;  2.  Plasmodium  malaria,  that 
of  quartan — cycle,  seventy-two  hours;  and  3.  Plasmodium  f aid  par  um,  that  of  aestivo- 
autumnal  or  malignant  tertian — cycle  of  forty-eight  hours. 

Multiple  Infections. — Variations  in  cycles  may  be  produced  by  infected  mosquitoes 
biting  on  successive  nights,J|p  that  one  crop  will  mature  and  sporulate  twenty-four 
hours  before  the  second.  Tr^is  would  give  a  quotidian  type  of  fever.  In  aestivo- 
autumnal  infections  anticipation  and  retardation  in  the  sporulation  cause  a  very 
protracted  paroxysm,  lasting  eighteen  to  thirty-six  hours;  this  tends  to  give  a  con- 
tinued or  remittent  fever  instead  of  the  characteristic  intermittent  type. 

Plasmodium  Vivax. — In  fresh,  unstained  preparations,  taken  at  the  time  of  the 
paroxysm  or  shortly  afterward,  the  benign  tertian  schizont,  or  nonsexual  parasite, 
is  seen  as  a  grayish  white,  round  or  oval  body,  whose  outlines  cannot  be  distinctly 
differentiated  from  the  infected  red  cell.  They  are  about  one-fifth  of  the  diameter 
of  the  red  cell  and  are  best  picked  up  by  noting  their  amoeboid  activity.  In  about 
eighteen  hours  fine  pigment  particles  appear  and  make  them  more  distinct.  After 
twenty-four  hours  the  lively  motion  of  the  pigment  and  the  projection  of  pseudopod- 
like  processes,  in  a  pale  and  swollen  red  cell,  makes  their  recognition  very  easy. 
When  about  thirty  to  thirty  ix  hours  old  the  amoeboid  movement  ceases. 
Approaching  the  merocyte  stage  the  pigment  tends  to  clump  into  one  or  two  pigment 
masses  and  one  can  recognize  small,  oval,  highly  refractile  bodies  within  the  sporulat- 
ing  parasite. 

The  gametes  or  sexual  forms  do  not  show  amoeboid  movement,  but  the  fully 
developed  gamete,  which  is  generally  larger  than  the  red  cells,  has  abundant  pig- 
ment, which  is  actively  motile  in  the  male  gamete  and  nonmotile  in  the  female. 
The  male  gamete  is  more  refractile,  is  rarely  larger  than  a  red  cell  and  shows  yellow 
brown,  short  rod-like  particles  of  pigment.  Abou^f teen  minutes  after  the  making 
of  a  fresh  preparation  these  male  gametes  "throw  out  four  to  eight  long,  slender, 
lashing  processes,  which  are  about  15  to  20  microns  long.  These  spermatozoon-like 
bodies  now  break  off  from  the  useless  parent  cell  and  with  a  serpent-like  motion  glide 
away  in  search  of  a  female  gamete,  knocking  the  red  cells  about  in  their  passage 
through  the  blood  plasma. 

The  female  gamete  is  larger  than  a  red  cell,  is  rather  granular  and  has  more  abi 
dant  dark  brown  pigment  than  the  male. 

Stained  Smears.— In  dried  smears,  stained  by  some  Romanowsky  method,  as  that 
of  Wright,  Leishman  or  Giemsa,  we  note  small  oval  blue  rings,  about  one-fifth  of  the 
diameter  of  the  infected  yellowish-pink  erythrocyte.  One  side  of  the  ring  is  dis 
tinctly  broader  than  the  rather  fine  opposite  end,  which  seems  to  hold  a  round, 
yellowish-brown  dot,  the  chromatin  dot,  and  has  a  resemblance  to  a  signet  ring. 
These  small  tertian  rings  of  the  nonsexual  parasites  (schizont)  are  seen  about  the 


288  THE   PROTOZOA 


vtotin 


time  of  the  commencement  of  the  sweating  stage  of  the  paroxysm.  Two  chromatin 
dots  in  the  line  of  the  ring  are  rare  as  is  also  true  of  more  than  one  ring  in  a  red  cell. 

When  the  parasite  is  about  twenty-four  hours  old  we  note  that  it  contains  much 
pigment  and  has  an  amoeboid  or  multiple  figure  of  eight  contour,  is  about  three- 
fourths  the  size  of  a  red  cell  and  that  the  infected  red  cell  is  about  one  and  one-half 
times  as  large  as  in  the  beginning  and  presents  a  washed-out  appearance.  It  is  an 
anaemic-looking  cell.  We  also  note,  as  characteristic  of  a  benign  tertian  infection, 
reddish-yellow  dots  in  the  pale  red  cell,  which  are  known  as  Schiiffner's  dots. 
These,  practically,  are  characteristic  for  benign  tertian. 

A  few  hours  before  the  completion  of  its  forty-eight-hour  cycle  the  contained 
pigment  begins  to  clump,  the  chromatin  to  divide  and,  finally,  we  have  a  sporulating 
parasite,  in  which  the  16  to  20  small,  round,  bluish  bodies,  with  chromatin  dots,  are 
irregularly  distributed  over  the  area  of  the  merocyte. 

The  gametes,  or  sexual  parasites,  show  a  thicker  blue  ring  and  have  the  chromatin 
dot  in  the  center  of  the  ring.  The  pigmentation  of  the  half-grown  gametes  is  more 
marked  than  that  of  schizonts  of  equal  size.  The  shape  of  the  gametes  is  not 
amoeboid,  as  is  that  of  the  twenty-four-  to  thirty-six-hour-old  schizont,  but  round 
or  oval.  The  full-grown  gametes  have  the  pigment  distributed  and  the  chromatin 
in  a  single  aggregation — -just  the  opposite  of  nonsexual  parasites.  The  male  gamete 
stains  a  light  grayish  blue  and  has  a  very  large  amount  of  chromatin,  usually  cen- 
trally placed.  The  female  gamete  stains  a  pure  blue,  has  only  about  one-tenth  as 
much  chromatin  as  plasma,  with  the  chromatin  often  placed  at  one  side.  The  pig- 
ment of  the  female  gamete  is  dark  brown  while  that  of  the  male  is  yellowish  brown. 

Plasmodium  Malariae. — In  fresh  preparations  the  young  quartan  schizont  has 
only  slight  amoeboid  movement  and,  as  development  proceeds,  the  rather  dark 
brown,  coarse  pigment  tends  to  arrange  itself  peripherally  about  the  band-shaped 
or  oval  parasite. 

The  infected  red  cell  shows  but  little  change.  At  the  end  of  seventy-two  hours 
the  rather  regular  daisy  form  of  the  merocyte  is  more  distinct  than  that  of  the  benign 
tertian  merocyte. 

The  distinctions  between  the  male  and  female  gametes  are  similar  to  those  of  the 
benign  tertian  gametes.  In  Romanowsky  stained  smears  it  is  difficult  to  distinguish 
the  young  quartan  schizont  from  the  benign  tertian  one  but,  after  twenty-four 
hours,  the  tendency  of  the  quartan  schizont  to  assume  equatorial  band  forms  across 
a  red  cell  of  normal  size  and  staining  characteristics  and  without  Schiiffner's  dots 
makes  the  differentiation  easy.  In  the  fully  developed  sporulating  parasite  or 
merocyte  the  eight  merozoites  assume  a  regular  distribution  giving  it  a  daisy 
appearance. 

The  gametes  show  practically  the  characteristics  of  the  benign  tertian  ones  but 
are  smaller. 

Plasmodium  Falciparum.— The  young  schizont  of  malignant  tertian  is  extremely 
difficult  to  detect  in  fresh  preparations,  there  being  noted  early  in  the  rather  long 
continued,  hot  stage,  only  small  crater-like  dots,  about  one-sixth  of  the  diameter  of 
a  red  cell  which,  however,  show  an  active  amoeboid  movement. 

Later  on  in  the  hot  stage  these  ring-like  dots  enlarge  to  become  about  one-third 
of  the  diameter  of  a  red  cell,  most  often  occupying  the  periphery  of  the  infected 
red  cell.  About  this  time,  or  at  the  very  commencement  of  the  pigmentation,  the 


CULTIVATION  OF  MALARIAL  PARASITE  289 

schizont  containing  red  cells  disappear  from  the  peripheral  circulation  so  that  the 
further  development  is  rarely  observed  in  blood  specimens. 

The  infected  cell  is  brassy  in  color  and  shrunken  in  shape — it  shows  evidences 
of  degeneration.  The  gametes  appear  as  crescent-shaped  bodies,  which  are  abso- 
lutely characteristic  of  malignant  tertian,  the  male  gamete  being  more  hyaline  and 
delicate  while  the  female  one  is  more  granular  and  larger. 

In  Romanowsky  stained  preparations  we  see,  while  the  fever  is  sustained,  small 
hair-like  rings,  with  geometrical  outline,  with  frequently  two  chromatin  dots  in  one 
end  of  the  ring  and  a  single  red  cell  often  showing  two  or  more  of  these  young  rings. 
The  rings  are  often  seen  as  if  plastered  on  the  periphery  of  the  red  cells  or  as  if  having 
destroyed  a  rounded  section  of  the  rim  of  the  red  cell.  As  the  fever  declines  the 
rings  tend  to  disappear  from  the  peripheral  circulation.  The  infected  red  cells  often 
show  polychromatophilia  and  distortion. 

In  old  aestivo-autumnal  cases,  or  those  with  severe  infection,  we  may  see  adult 
rings  and  merocytes,  which  latter  are  smaller  than  those  of  benign  tertian,  show  from 
1 6  to  32  irregularly  placed  merozoits  and  a  sharply  clumped  mass  of  pigment. 

The  gametes  are  the  striking  crescent-shaped  bodies  and  these  show  the  distinc- 
tions of  blue  staining  for  the  female,  with  lighter  gray-green  staining  and  abundance 
of  chromatin  for  the  male.  The  chromatin  staining  of  crescents  does  not  stand  out 
so  well  as  that  of  the  round  form  gametes  of  benign  tertian  and  quartan. 

As  regards  differentiation  of  species  and  cycle  the  examination  of 
stained  smears  is  more  satif  actory  and  definite,  as  well  as  less  time  con- 
suming. Still,  one  obtains  many  points  of  differentiation  in  the  fresh 
preparation  and  should  study  such  a  preparation  while  carrying  out  the 
staining  of  his  dried  smear. 

Central  vacuolation  of  red  cells  is  common  in  malarial  anaemia  and  may  be  mis- 
taken for  nonpigmented  parasites. 

Malarial  rings  are  usually  peripheral  and  do  not  vary  in  size  as  one  focuses  up  and 
down  as  do  the  central  vacuoles. 

A  very  puzzling  but  well-recognized  finding  in  cases  treated  with  quinine  or  sal- 
varsan  is  the  so-called  quinine  affected  parasite.  Such  parasites  lack  definiteness 
of  outline  and  show  poor  chromatin  staining.  The  gametes  do  not  seem  to  show 
these  effects  from  the  drug. 

Cultivation. — As  to  cultivation  of  malarial  parasites.  Bass  takes  from  10  to  20 
c.c.  of  blood  from  the  malarial  patient's  vein  in  a  centrifuge  tube  which  contains 
Ho  c.c.  of  50%  glucose  solution.  A  glass  rod,  or  a  piece  of  tubing  extending  to 
the  bottom  of  the  centrifuge  tube  is  used  to  defibrinate  the  blood.  After  centri- 
fugalizing  there  should  be  at  least  i  inch  of  serum  above  the  cell  sediment.  The 
parasites  develop  in  the  upper  cell  layer,  about  >£0  to  Ko  inch  from  the  top.  All  of 
the  parasites  contained  in  the  deeper  lying  red  cells  die.  To  observe  the  develop- 
ment, red  cells  from  this  upper  Ko-inch  portion  are  drawn  up  with  a  capillary  bulb 
pipette. 

Should  the  cultivation  of  more  than  one  generation  be  desired,  the  leukocyte  uppe 
layer  must  be  carefully  pipetted  off,  as  the  leukocytes  immediately  destroy  the 
merozoites.     Only  the  parasites  within  red  cells  escape  phagocytosis. 


290 


THE   PROTOZOA 


parasites  are  much  more  resistant.  Bass  thinks  he  observed  parthenogen 
The  temperature  should  be  from  40°  to  4i°C.  and  strict  anaerobic  conditions  ob- 
served. ^Estivo-autumnal  organisms  are  more  resistant  than  benign  tertian  ones. 
Dextrose  seems  to  be  an  essential  for  the  development  of  the  parasites. 

Bass  considers  that  P.  vivax  has  a  disc-like  structure  which  enables  it  to  squeeze 
through  the  brain  capillaries  while  adult  schizonts  of  P.  falciparum  have  a  solid 
oval  form  which  causes  them  to  be  caught  in  the  capillaries. 

The  Thompsons  have  rather  .simplified  the  method  of  Bass.  They  draw  10  c.c. 
of  blood  into  a  test-tube  containing  the  usual  amount  of  glucose  solution.  They 
then  defibrinate  the  blood  by  stirring  with  a  thick  wire  for  about  five  minutes  and 
remove  the  wire  with  the  adhering  clot.  They  then  pour  this  defibrinated  blood 
into  several  small  sterile  test-tubes,  which  should  contain  at  least  a  i-inch  column. 
Rubber  caps  are  adjusted  over  the  cotton  plugs  and  the  tubes  placed  in  the  incu- 
bator. They  note  the  tendency  of  cultures  of  P.  falciparum  to  agglutinate  which 
is  not  true  of  P.  vivax. 

They  think  this  agglutination  the  great  cause  of  the  plugging  of  capillaries  in 
pernicious  malaria.  They  note  32  merozoites  as  maximum  number  in  sporulation 
of  P.  falciparum  while  P.  vivax  has  usually  16  or  more,  but  never  as  many  as  32 

This  would  explain  the  shorter  incubation  period  of  malignant  tertian.  The 
pigment  of  P.  falciparum  clumps  much  earlier  in.  the  developing  schizont  than  that 
of  P.  vivax  and  is  much  coarser  and  more  discrete. 

While  Bass  thought  he  noted  parthenogenesis  in  cultures  others  have  failed  to 
observe  any  evidence  of  it. 


UNSTAINED  SPECIMEN  (FRESH  BLOOD) 


P.    vivax 
(benign  tertian) 

P.    malariae 
(quartan) 

P.  falciparum 
(malignant  tertian) 
(aestivo-autumnal) 

Character  of  the 
infected  red  cell. 

Swollen  and  light  in 
color  after  eighteen 
hours. 

About  the  size  and 
color  of  a  normal  red 
cell. 

Tendency  to  distortion  of 
red  cell  rather  than  crena- 
tion.  Shriveled  appear- 
ance. (Brassy  color.) 

Character  of  young 
schizont. 

Indistinct  amoeboid 
outline.  Hyaline. 
Rarely  more  than 
one  in  r.c.  Active 
amoeboid  move- 
ment. One-third 
diam.  of  r.c. 

Distinct  frosted  glass 
disc.  Very  slight 
amoeboid  motion. 

Small,  distinctly  round, 
crater-like  dots  not  more 
than  one-sixth  diameter 
of  red  cell.  Two  to  four 
parasites  in  one  red  cell 
common.  Shows  amoe- 
boid movement  until  ap- 
pearance of  pigment. 

Character    of    ma- 
ture schizont. 

Amoeboid  outline. 
No  amoeboid  move- 
ment. 

Rather  oval  in  shape. 
Sluggish  movement 
of  peripherally 
placed  coarse  black 
pigment. 

Only  seen  in  overwhelming 
infections.  Have  scanty 
fine  black  pigment 
clumped  together. 

Pigment. 

Fine  yellow-brown, 
rod-like  granules 
which  show  active 
motion  in  one-half- 
grown  schizont. 
Motion  ceases  in 
full-grown  schizont. 

Coarse  almost  black 
granules.  Shows 
movement  only  in 
young  to  half-grown 
schizont. 

Pigmented  schizonts  very 
rare  in  peripheral  circula- 
tion except  in  overwhelm- 
ing infections.  Tends  to 
clump  as  eccentric  pig- 
ment masses  almost  black 
in  color. 

PIROPLASMS 
STAINED  SPECIMEN 


291 


P.   vivax 
(benign   tertian) 

P.   malarias 
(quartan) 

P.  falciparum 
(malignant  tertian) 
(aestivo-autumnal) 

Character  of  in- 
fected red  cell. 

Larger  and  lighter 
pink  than  normal 
red  cell.  Shows 
"Schuffner's  dots." 

About  normal  size 
and  staining. 

Shows  distortion  and  some 
polychromatophilia  and 
stippling.  Rarely  we 
have  coarse  cleft  -like  red- 
dish dots—  Maurer's  spots. 

Character  of  young 
schizont. 

Chromatin  mass  usu- 
ally single  and  situ- 
ated in  line  with  the 
ring  of  the  irregularly 
outlined  blue   para- 
site. 

Rather  thick  round 
rings  which  soon 
tend  to  show  as 
equatorial  bands. 

Very  small  sharp  hair-like 
rings,  with  a  chromatin 
mass  protruding  from  the 
ring.  Often  appears  on 
periphery  of  red  cell  as  a 
curved  blue  line  with 
prominent  chromatin  dot. 
Frequently  two  chro- 
matin dots. 

Character  of  half- 
grown  schizont. 

Vacuolated  or  Fig.  8 
l9op-like  body  with 
single  chromatin  ag- 
gregation. Schuff- 
ner's dots. 

More  marked  band 
forms  stretching  ac- 
ross r.b.c. 

Not  often  found  in  periph- 
eral circulation.  C  h  r  o  - 
matin  still  compact. 

Character  of  ma- 
ture schizont. 

Fine  pigment  rather 
evenly  distributed 
in  irregularly  out- 
lined parasite. 

Coarse  pigment 
rather  peripherally 
arranged  in  an  oval 
parasite. 

Very  rarely  seen  in  periph- 
eral circulation  in  ordi- 
nary infection.  Pigment 
clumps  early. 

Character  of  mero- 
cyte. 

Irregular  division  into 
15    or    more    spore- 
like   chromatin    dot 
segments. 
Mulberry. 

Rather  regular  di- 
vision into  eight  or 
10  merozoites  — 
Daisy. 

Sporulation  occurs  in 
spleen,  brain,  etc.  Rarely 
in  peripheral  circulation. 
Eight  to  10  chromatin 
staining  merozoites. 
(In  culture  32.) 

Character  of  mac- 
rogamete. 

Round  deep  blue. 
Abundant,  rather 
coarse  pigment, 
chromatin  at  per- 
iphery. 

Round,  similar  to  P. 
vivax  but  smaller. 

Crescentic,  deep  blue,  pig- 
ment clumped  at  center, 
chromatin  scanty  and  in 
center. 

Character  of  mi- 
crogametocyte: 

Round,  light  green- 
blue,  pigment  less 
abundant,  c  h  r  o  - 
matin  abundant  and 
located  centrally  or 
in  a  band. 

Round  like  P.  vivax. 

More  sausage-shaped  than 
crescent.  Light  blue. 
Pigment  scattered 
throughout.  Chromatin 
scattered  and  in  greater 
quantity  but  difficult  to 
stain. 

PIROPLASMS 

Belonging  like  the  malarial  parasite  to  the  Haemosporidia  we  have  a 
group  of  parasites  known  as  the  PIROPLASMS.  The  correct  name  for 
these  parasites  is  Babesia  but  they  are  better  known  under  the  name 
Piroplasma.  They  are  minute  organisms,  usually  pear  or  rod  shape, 
which  invade  the  red  corpuscles.  They  produce  no  pigment  but 
destroy  the  corpuscle  and  set  free  the  Hb.  which  is  excreted  in  enor- 
mous amounts  by  the  kidneys.  It  is  this  which  gives  the  name  red- 
water  to  the  disease  usually  designated  Texas  fever  of  cattle. 


2Q2 


THE    PROTOZOA 


The  cause  of  this  disease  is  B.  bovis  (B.  bigemina)  and  the  parasite  is  transmit 
by  a  tick,  Mar  gar  opus  annulatus.  There  is  also  a  disease  of  dogs  called  malignant 
jaundice  of  dogs  which  is  caused  by  B.  canis  and  also  transmitted  by  a  tick.  Organ- 
isms of  this  kind  have  been  thought  of  in  connection  with  blackwater  fever  of  man. 
Seidelin  has  claimed  that  a  parasite  of  similar  nature,  Paraplasma  flavigenum,  was 
the  cause  of  yellow  fever. 

At  one  time  spotted  fever  of  the  Rocky  Mountains  was  supposed  to  be  due  to  a 
parasite  named  Babesia  hominis.  The  parasite  of  Oroya  fever  is 
probably  protozoal  in  nature.  It  is  found  in  red  cells  as  rod- 
like  bodies  and  is  called  Bartonella  bacllliformis .  See  Oroya 
fever. 

SARCOSPORIDIA 

Sarcosporidia  are  sporozoa  found  in  the  striped 
muscles  of  various  mammals  and  birds.  They  are 
common  in  the  pig  and  mouse  and  have  been  re- 
ported for  man  in  three  well-authenticated  cases.  In 
the  last,  Darling  found  these  protozoa  in  the  biceps 
muscle  of  a  negro  patient  in  Panama.  In  Baraban's 
case  the  laryngeal  muscles  at  autopsy  were  found 
to  show  cysts  about  Jf  5  inch  long  which  contained 
sickle-shape  sporozoites  about  qn  long. 

FIG.  63. — Mie-  They  are  known  also  as  Miescher's  tubes  when  in  muscle  fibers, 
scher's  sac  from  They  are  divided  into  three  genera:  Miescheria  and  Sarcocystis 
olTa  'ho501*1  XU1Q  when  Parasitic  "*  muscle  fiberJ  Balbiania,  when  parasitic  in  the 
diameters.  (After  intervening  connective  tissue  of  the  muscles.  The  method  of 
Ostertag.)  transmission  is  unknown.  In  some  places  more  than  50%  of 

the  sheep  and  pigs  may  show  infection. 

Miescheria  has  a  thin  membrane  surrounding  the  cyst  while  that  of  Sarcocyslis 
is  thickened  and  radially  striated  by  small  canaliculi. 

As  the  young  trophozoite  grows  nuclei  increase  and  a  definite  membrane  forms 
which  the  sporoblasts  eventually  fill.  According  to  Minchin  the  Sarcosporidia  con- 
tain only  one  genus,  Sarcocystis.  It  is  never  parasitic  for  invertebrate  hosts  and 
while  occasionally  found  in  birds  and  reptiles  it  is  preeminently  a  parasite  of  the 
higher  vertebrates.  As  a  rule,  they  are  harmless  parasites  but  the  Sarcocystis  muris 
is  very  pathogenic  for  the  mouse.  Closely  related  to  the  order  Sarcosporidia  is  the 
parasite  Rhinos poridium  kinealyi. 

Rhinosporidium  kinealyi. — It  causes  pedunculated  tumors  of  nasal  cavity.  The 
pansporoblasts  enlarge  in  the  center  of  the  connective  tissue  of  the  nasal  polyp  and 
contain  about  12  sporoblasts.  When  mature  the  cystic-like  polyp  bursts  and  the 
sporoblasts  are  liberated  to  extend  the  infection. 


CELL  INCLUSION  DISEASES  293 

CHLAMYDOZOA 

These  organisms  are  generally  considered  as  being  protozoal  in 
nature  and  as  a  rule  belong  to  the  filterable  viruses,  which  is  the  desig- 
nation for  the  infectious  principles  of  those  diseases,  in  which  filtration 
of  defibrinated  blood  or  serum  through  a  Berkefeld  filter  capable  of 
holding  back  so  small  an  organism  as  the  M.  melitensis,  does  not  pre- 
vent the  infection  being  transmitted  when  introduced  by  the  proper 
atrium  of  infection.  The  Chlamydozoa  are  also  characterized  by  the 
occurrence  of  "cell  inclusions." 

The  best  known  infections  of  this  group  of  diseases  in  man  are  smallpox,  vaccinia, 
rabies,  trachoma,  molluscum  contagiosum,  and  foot  and  mouth  disease.  There  are 
many  such  infections  in  other  animals.  The  cell  inclusions  are  regarded  as  prod- 
ucts of  cellular  reaction  to  a  virus  which  is  more  or  less  impossible  of  demonstra- 
tion. The  discovery  of  exceedingly  minute  granules  in  some  of  these  diseases,  as 
in  variola  and  trachoma,  has  suggested  that,  as  a  reaction  to  the  invasion  by  such 
a  granule,  the  cell  throws  an  eveloping  mantle  about  the  invading  particle.  To 
designate  this  we  use  the  name  Chlamydozoa. 

The  generic  name  Cytorrhyctes  has  been  applied  to  certain  of  these  viruses,  thus 
C.  vaccinia  develops  within  the  epithelial  cells  of  stratified  epithelium.  In  vaccinia, 
Councilman  and  his  colleagues  consider  that  the  development  only  takes  place  in 
the  cytoplasm  of  the  cell.  In  variola,  however,  the  developmental  cycle  affects  the 
nucleus. 

Cytorrhyctes  luis,  reported  as  the  cause  of  syphilis,  sporulates  in  the  blood-vessels 
and  in  the  connective  tissue,  not  in  epithelial  cells. 

Cytorrhyctes  scarlatina  was  reported  by  Mallory  to  have  been  found  in  the  skin 
in  four  cases  of  scarlet  fever. 


CHAPTER  XVII 


FLAT  WORMS 

CLASSIFICATION  OF  THE  PLATYHELMINTHES  (FLAT  WORMS) 


Class 


Family 


Trematoda 


Cestoda 


Fasciolidae 


Paramphistomidae 


Schistosomidae 


Dibothriocephalidae 


Tamiidae 


Genus 

Species 

Fasciola 
Fascioletta 

F.  hepatica 
F.  ilocana 

Fasciolopsis 
Dicroccelium 

F.  buski 
D.  lanceatum 

Paragonimus 
\   Opisthorchis 

Clonorchis 

Heterophyes 
Cladorchis 

P.  westermanii 
O.  felineus 
(C.  sinensis 
C.  endemicus 
H.  heterophyes 
C.  watsoni 

Gastrodiscus 

G.  hominis 

{S.   haematobium 

Schistosoma 

S.  japonicum 
S.   mansoni 

f  Dibothriocephalus 
\   Diplogonoporus 
Dipylidium  - 

Hymenolepis 

D.  latus 
D.  grandis 
D.  caninum 
H.  nana 
H.  diminuta 

Taenia 
Davainea 

T.  solium 
T.  saginata 
D.  madagascariensis 

NOTE. — Two  larval  Taeniidae  are  found  in  man  (Cysticercus  celluloses  and  Echino- 

coccus  polymorphous). 

Also  two  larval  Dibothriocephalidae  (Sparganum  mansoni  and  Sparganum  prolifer.) 
Two  parasites  often  referred  to  as  ophthalmic  flukes  have  been  reported  lying 

between  the  crystalline  lens  and  its  membrane.     They  have  been  considered  as 

possibly  trematode  larvae.     Distomum  ophlhalmobium  was  found  in  1850  in  the  eye 

of  a  child  and  Monostoma  lentis  in  the  eye  of  an  old  woman. 

TREMATODES  OR  FLUKES 

Flukes  are  generally  leaf-like  in  outline,  rarely  cylindrical,  and  ex- 
hibit marked  variation  in  size  and  shape.     They  are  nonsegmented  and 

294 


FLUKES 


295 


do  not  have  cilia  on  ectoderm.  Very  characteristic  of  them  is  the 
possession  of  suckers  by  which  they  hold  on  to  the  skin  or  alimentary 
system  of  their  host. 


3*3 


They  are  divided  into  two  orders:  i.  the  Monogenea  in  which  the  egg  gives  rise 
to  a  larva  which  later  becomes  the  adult  and  2.  the  Digenea.  It  is  to  this  latter  that 
the  flukes  parasitic  in  man  belong.  This  order  is  characterized  by  the  fact  that  the 
larva  becomes  parasitic  in  some  second  animal  and  then  gives  rise  to  a  second  gen- 
eration of  larvae  which  latter  develop  into  adults. 


296  FLAT   WORMS 

The  largest  human  fluke,  Faciolopsis  buskl,  is  from  2  to  3  inches  (50  to 
75  mm.)  in  length,  while  the  Heterophyes  heterophyes  is  less  than  ^2  inch  (2 
mm.)  in  length.  The  most  important  fluke,  the  liver  fluke,  Clonorchis  endemicus, 
is  flat  and  almost  transparent,  while  the  almost  equally  important  lung  fluke,  the 
Paragonimus  westermanii,  is  oval,  almost  round  and  reddish  brown  in  color.  With 
the  exception  of  the  Schistosomidae,  all  flukes  are  hermaphrodites,  and,  with  the 
exception  of  this  family,  all  flukes  have  operculated  eggs.  The  only  other  opercu- 
lated  (with  a  lid)  eggs  we  meet  with  in  man  are  those  of  the  Dibothriocephalidse. 

The  three  important  families  of  flukes  parasitic  for  man  are: 

1.  Paramphistomidae — flukes  with  two  suckers  situated  at  either 
extremity. 

2.  Fasciolidae — flukes  with  two  suckers,  one  terminal,  the  other  ad- 
jacent to  it  and  situated  ventrally.     This  family  includes  the  im- 
portant genera  Fasciola,  Opisthorchis,  Dicroccelium,  Fasciolopsis,  and 
Paragonimm.     In  Paragonimus   and   Heterophyes  the  genital  pore  is 
posterior  to  the  acetabulum,  in  the  other  genera  it  is  anterior.    Fas- 
ciola  has  a  dendritic  intestinal  canal  which  is  not  the  case  with  Clon- 
orchis,    Fasciolopsis,     Fascioletta,     Opisthorchis     and     Dicroccelium. 
In  Dicrocxlium  the  testicles  are  anterior  to  the  uterus,  in  Opisthorchis, 
Clonorchis,  Fasciolopsis  and  Fasciohtta  they  are  posterior.    Fascio- 
lopsis and  Clonorchis  have  branched  testicles  (the  former  a  very  large 
ftuke-Clonorchis  of  medium  size)  while  those  of  Opisthorchis  are  lobed. 

3.  Schistosomidae:  In  this  family  we  have  a  leaf -like  male  which  by 
a  folding  in  of  its  sides  makes  a  channel  for  the  thread-like  female.     The 
sexes  are  separate,  not  hermaphroditic  as  with  the  Fasciolidae  and 
Paramaphistomidae. 

Flukes  have  two  suckers  which,  except  in  the  Paramphistomidae,  are  quite  near 
each  other — one  is  termed  the  oral  sucker  and  the  other  the  ventral  sucker  or  acet- 
abulum. The  intestinal  tract  consists  of  a  pharynx,  proceeding  from  the  oral  sucker 
which  bifurcates  and  terminates  in  blind  intestinal  caeca. 

At  the  posterior  extremity  is  an  excretory  pore  which  is  at  the  termination  of 
a  duct  which  divides  into  ramifying  branches.  This  is  the  water-vascular  system. 
The  testes,  of  various  shapes  and  relations  to  the  uterus,  are  more  or  less  centrally 
situated  and  have  vasa  deferentia.  In  some  flukes  the  receptaculum  seminis  is  a 
conspicuous  organ.  The  vitellaria  are  bilateral  branching  glands  which  pour 
nutrient  material  into  the  ootype.  It  is  in  the  ootype  that  the  eggs  are  formed, 
and  opening  into  it  we  have  the  adjacent  ovary.  The  shell  gland  is  near  the  ovary. 

A  canal,  known  as  Laurer's  canal,  leads  from  the  ootype  to  the  exterior,  the 
function  of  which  is  in  question.  It  is  probable  that  as  trematodes  have  no  sperma- 
theca,  the  spermatozoa  from  other  flukes  enter  by  way  of  this  canal.  The  life  his- 
tdry  of  the  important  human  flukes  is  unknown.  It  is  supposed  that  this,  in  a  meas- 
ure, may  resemble  that  of  the  common  liver  fluke  of  sheep  (sheep  rot).  In  this  the 
eggs  containing  a  ciliated  embryo  (miracidium)  pass  out  in  the  faeces.  This  embryo 


LIVER  FLUKES  2Q7 

is  hatched  out  and,  gaining  the  water,  swims  about  actively  until  it  reaches  some 
suitable  mollusk  (Limnaa  truncatula) .  By  means  of  a  pointed  end,  it  bores  its 
way  into  the  body  of  the  gastropod  and  in  the  pulmonary  chamber  becomes  a 
bag-like  structure  (the  sporocyst)  from  the  germinal  cells  of  which  develop  a  creature 
with  an  alimentary  canal  (redia).  The  rediae  tend  to  break  out  of  the  sporocyst  and 
wander  to  the  liver  of  the  snail.  These  rediae  may  give  rise  to  a  second  generation 
of  rediae. 

From  the  rediae  minute  little  worms  resembling  adult  flukes  in  possessing  suckers, 
but  differing  in  the  possession  of  a  tail,  develop  (cercaria)..  Having  reached  maturity, 
these  cercariae  leave  the  rediae,  and,  as  in  case  of  Faseiola  hepatica,  lose  the  tail,  be- 
come encysted  on  blades  of  grass,  to  be  eaten  by  sheep  and  again  commence  the  cycle. 
The  encysted  cercariae  develop  into  adult  liver  flukes.  It  is  probable  that  with  many 
flukes  the  cercariae  enter  some  host,  as  mollusk,  insect,  or  fish,  and  that  it  is  by  eat- 
ing such  animals  as  food  that  man  becomes  infected.  Looss  thinks  it  possible  that 
the  miracidium  of  Schistosoma  hcematobium  may  bore  its  way  directly  into  man, 
as  do  the  larvae  of  the  hookworm.  Manson  also  suggests  that  the  reporting  by  Mus- 
grave  of  100  mature  lung  flukes  in  a  psoas  abscess  makes  it  very  probable  that  these 
parasites  entered  the  body  as  miracidia.  The  idea  in  China  is  that  the  infection 
with  the  common  liver  fluke  of  man  is  brought  about  by  eating  fish.  Fluke  disease 
is  generally  known  as  distomatosis  or  distomiasis. 

LIVER  FLUKES 

Faseiola  hepatica  (Dislomum  hepaticum). — This  fluke,  while  of 
enormous  economic  importance  by  reason  of  destruction  of  sheep,  has 
only  been  reported  23  times  in  man,  and  in  these  instances  does  not 
seem  to  have  occasioned  marked  symptoms. 

It  has  a  cone-shaped  anterior  projection  and  is  about  1%  inches  (30  mm.)  long. 
The  intestinal  canal,  as  well  as  the  testicles,  is  branched.  These  intestinal  diver- 
ticula  are  well  marked  in  the  cone  just  after  the  branching  from  the  oesophagus. 
Diameter  of  acetabulum  about  1.6  mm.,  of  oral  sucker  i  mm.  There  is  a  possible 
importance  of  F.  hepatica  in  connection  with  a  peculiar  affection  known  as 
"halzoun."  This  results  from  the  eating  of  raw  goat-liver,  and  it  is  supposed  that 
the  flukes  crawl  up  from  the  stomach  and,  entering  the  larynx  or  attaching  themselves 
about  the  glottis,  produce  the  asphyxia  characteristic  of  the  disease.  ^ 

Dicrocoslium  lanceatum.— This  has  only  been  reported  seven  times  in  man. 
The  symptoms  are  unimportant.  The  fluke  is  about  Yz  inch  (8  mm.)  long,  with 
testicles  anterior  to  the  uterus. 

Clonorchis  endemicus  (Opisthorchis  sinensis).— This  fluke  and  the 
C.  sinensis  are  the  most  important  of  the  human  liver  flukes.  Until 
recently  these  flukes  were  known  as  Opisthorchis  sinensis. 

Looss  has  separated  this  genus  from  Opisthorchis  principally  by  the  character- 
istic of  branching  testicles— those  of  Opisthorchis  being  lobed.  This  fluke  is  very 
common  in  China  and  Japan— in  certain  sections  of  Japan  20%  of  the  population 


298  FLAT    WORMS 

being  infected.  This  fluke  is  about  y±  to  }£  inch  (8  mm.)  long  and  C.  sinensis  about 
%  inch  long  and  K  inch  broad  (16  X  4  mm.).  When  squeezed  out  of  the 
thickened  bile  ducts  it  is  so  transparent  and  glairy  as  almost  to  resemble  glairy 
mucus.  As  many  as  4000  of  these  parasites  have  been  found  in  a  case,  chiefly  in 
the  liver,  but  at  times  in  the  pancreas.  This  fluke  is  supposed  to  produce  most 
serious  symptoms,  as  indigestion,  swelling  and  tenderness  of  liver,  ascites,  oedema, 
and  a  fatal  cachexia.  As  a  matter  of  fact,  many  physicians  in  China  attribute  very 
little  pathogenic  importance  to  it.  The  disease  is  diagnosed  by  the  presence  of  the 
ova  in  the  stools.  The  source  of  infection  is  probably  through  the  eating  of  uncooked 
fish. 

Kobayashi  has  examined  various  mollusks  and  fish  for  trematode  larvae.  He 
succeeded  in  infecting  nine  kittens  and  two  cats  by  feeding  them  with  certain  fresh- 
water fishes  whose  flesh  contained  trematode  larvae.  These  fish  were  found  in 
districts  where  human  distomiasis  was  common.  The  view  is  taken  that  the  two 
species  of  Clonorchis  are  identical. 

Opisthorchis  felineus. — This  fluke  is  smaller  than  the  C.  endemicns,  and  is  a 
common  parasite  of  the  gall-bladder  and  bile  ducts  of  cats.  There  are  two-lobed 
testicles  in  this  species  instead  of  dendritic  ones  as  in  C.  endemicus.  In  certain 
parts  of  Siberia  the  parasite  is  found  in  more  than  6%  of  the  human  autopsies.  The 
symptoms  are  similar  to  those  caused  by  C.  endemicus. 

Other  liver  flukes  of  less  importance  which  have  been  reported  for  man  are:  i. 
Opisthorchis  noverca.  This  was  found  in  bile  ducts  of  two  natives  of  Calcutta.  It 
was  lancet-shaped  and  covered  with  spines. 

2.  Metorchis  truncatus:  This  is  a  small  fluke,  K2  incn  (2  mm.)  long,  squarely 
cut  across  at  Its  posterior  end  and  covered  with  spines.  This  was  possibly  found 
once  in  man. 

Intestinal  Flukes 

Cladorchis  watsoni  (Amphistomum  watsoni). — This  fluke  is  about  ^  inch 
(8  mm.)  long,  of  oval  outline  but  broader  at  posterior  end  and  has  an  indistinct  oral 
sucker  and  a  large  sucker  at  the  other  end.  This  parasite  has  been  reported  from 
northern  Nigeria  and  is  said  to  be  a  common  infection  of  regions  about  Lake  Chad. 
Eggs,  125  X  75M- 

Gastrodiscus  hominis  (Amphistomum  hominis). — This  fluke  is  about  ^  inch 
(6  mm.)  long  and  has  a  disc-like  concavity,  about  Y§  inch  in  diameter  from 
which  proceeds  a  teat-like  projection,  bearing  an  oral  sucker.  The  acetabulum 
is  in  the  posterior  border  of  the  disc.  While  it  has  only  been  reported  a  few  times  for 
man,  indications  are  that  it  is  probably  fairly  common  in  India  and  Assam. 
Eggs,  150  X  72/x.  It  gives  rise  to  dysenteric  symptoms. 

Fasciolopsis  buski  (Distomum  crassum). — This  is  probably  a  rather  common 
parasite  in  India,  as  Dobson  found  the  eggs  in  i%  of  the  stools  of  more  than 
1000  coolies.  The  fluke  is  from  2  to  3  inches  (40  to  70  mm.)  in  length  and  about  ^ 
inch  (12  mm.)  in  breadth.  It  is  thick,  brown  in  color,  and  has  a  very  large  acetabu- 
lum, three  times  the  size  of  the  oral  sucker  and  located  almost  adjacent  to  it.  The 
branched  ovary  and  shell  gland  lie  in  the  center  with  the  branched  testicles  posterior. 
The  coiled  uterus  is  anterior  to  the  testicles.  Eggs,  125  X  75M-  These  parasites 
cause  dyspeptic  symptoms  and  an  irregular  diarrhoea.  It  is  also  called  Distomum 


LUNG  FLUKES  299 

crassum.  F.  rathouisi  is  now  considered  to  have  been  a  shrunken  F.  buski,  as  it 
seems  to  be  anatomically  similar  to  F.  buski,  Kwan's  fluke  reported  from  Hong 
Kong,  was  possibly  F.  buski. 

Heterophyes  heterophyes  (Cotylogonimus  heterophyes).— This  exceedingly 
small  fluke  (2  X  0.5  mm.),  which  can  be  recognized  by  its  small  size  (less  than  ^2 
inch  long)  and  large,  prominent  acetabulum,  was  formerly  supposed  to  be  rare. 
The  oral  sucker  is  much  smaller  than  the  acetabulum.  The  elliptical  testicles  lie 
at  the  extreme  posterior  end.  Cuticle  has  scale-like  spines.  The  eggs  are  30  X  I7M- 
Very  characteristic  of  this  genus  is  the  large  sucker-like  genital  pore  just  below  and 
to  one  side  of  the  acetabulum.  Looss  has  shown  that  it  is  quite  common  in  Egypt, 
he  having  found  it  twice  in  Alexandria  in  nine  autopsies.  The  parasites  occupy  the 
ileum.  It  is  common  in  dogs. 

Echinostoma  ilocana  (Fascioletta  ilocana).— This  is  a  small  fluke,  about  Y±  inch 
(6  mm.)  long.  There  are  two  massive  testicles  in  the  posterior  part  of  body.  The 
acetabulum  is  prominent  and  about  5ooju  in  diameter.  This  fluke  has  a  ring  of 
spines  around  the  anterior  extremity.  Ovary  anterior  to  testes.  Genital  pore 
anterior  to  acetabulum.  The  egg  of  this  small  fluke  is  quite  large  (ioo/i)  and  has  an 
operculum.  These  trematodes  were  found  by  Garrison  in  five  natives  of  Luzon, 
P.  I.,  after  treatment  with  male  fern. 

LUNG  FLUKES 

Paragonimus  westermanii  (Distoma  ringeri). — In  certain  parts  of 
Japan  and  Formosa  it  is  estimated  that  as  many  as  10%  of  the  inhab- 
itants may  harbor  this  parasite. 

It  is  also  common  in  China,  and  recently  many  cases  have  been  reported  in  the 
Philippines.  Dr.  Stiles  states  that  around  Cincinnati,  Ohio,  there  was  at  one  time 
quite  a  heavy  infection  among  the  hogs,  so  that  it  may  be  that  certain  cases  diagnosed 
in  man  as  pulmonary  tuberculosis  are  paragonimiasis. 

It  is  popularly  known  as  endemic  haemoptysis  on  account  of  the  accompanying 
symptoms  of  chronic  cough  and  expectoration  of  a  rusty-brown  sputum.  After 
violent  exertion,  and  at  times  without  manifest  reason,  attacks  of  haemoptysis  of 
varying  degrees  of  severity  come  on.  The  characteristic  ova  are  constant  in  the 
sputum  and  establish  the  diagnosis.  The  fluke  itself  is  a  little  more  than  Y$ 
inch  (8  mm.)  long  and  is  almost  round  on  transverse  section,  there  being,  however, 
some  flattening  of  the  ventral  surface.  The  acetabulum  is  conspicuous  and  opens 
just  anterior  to  the  middle  of  the  ventral  surface.  Eggs  about  90  X  6s/z. 

The  branched  testicles  are  posterior  to  the  laterally  placed  uterus  and  the  genital 
pore  opens  below  the  acetabulum.  The  branched  ovary  is  opposite  the  uterus  on  the 
other  side. 

It  is  rather  flesh-like  in  appearance  and  is  covered  with  scale-like  spines.  The 
flukes  are  usually  found  in  tunnels  in  the  lungs,  the  walls  of  which  are  of  thickened 
connective  tissue.  There  may  be  also  cysts  formed  from  the  breaking  down  of 
adjacent  tunnel  walls.  In  addition  to  lung  infection  with  this  fluke,  brain,  liver, 
and  intestinal  infections  may  be  found.  Musgrave  was  the  first  one  to  call  attention 
to  the  frequency  of  general  infection  with  this  parasite  (paragonimiasis)  in  the 


300 


FLAT   WORMS 


J     4-U~ 


Philippines.     He  found  it  in  17  cases  in  one  year.     The  life  history,  beyond  the 
the  stage  of  miracidium,  is  unknown. 

Another  fluke  which  has  been  reported  from  the  lung  is  Fasciola  gigantea  (very 
similar  to  F.  hepatica).  This  was  coughed  up  by  a  French  officer  who  had  been  in 
Africa. 


Ova  of  the  Parasitic  Worms  or  Man 
TREMATODA 


DRAWN  TO  S    C     A    L    C  X       IOOO 


Heterophyes 
heterophyes 


Dicro 
coelium 
lance  utum 

P«»~*4     Fasciola 

*_  — 


Fasciolopsis 


Opisthorchis 
felineus  ( 


Clqiiorcliis  Clonorchis 
sinensis  endemicus 

iMudiix-il  fioiii  Looss  1(K>;) 


n  i 

//J; 


Schistosoma 


FIG.  66. — Trematode  ova. 


BLOOD  FLUKES 

Schistosoma  haematobium. — Flukes  of  the  circulatory  system  are 
of  great  importance  in  Egypt,  South  Africa,  Japan,  and  the  West 
Indies.  The  disease  is  named  bilharziasis  after  Bilharz  who  in  1851 
first  associated  the  parasite  and  the  disease. 

It  seems  probable  that  there  are  at  least  three  human  species, 
differentiated  principally  by  the  appearance  of  the  egg.  In  the  blood- 
fluke  disease  of  Egypt  (S.  hamatobium) ,  the  parasite  chiefly  infects 
the  bladder  and  the  egg  has  a  terminal  spine.  The  terminal-spined 
ovum  is  also  found  in  the  rectum  and  in  the  faeces.  In  the  West 


BLOOD  FLUKES 


301 


Indies,  as  shown  by  the  reports  of  Surgeon  Holcomb  from  Porto  Rico, 
rectal  bilharziasis  is  rather  common.  In  these  cases  the  egg  is  prac- 
tically always  lateral-spined.  Looss  thinks  that  the  lateral-spined  egg 
is  the  product  of  an  unfertilized  female  S.  hamatobium.  These  flukes 
differ  from  other  human  flukes  in  possessing  nonoperculated  eggs  as 
well  as  in  having  the  sexes  separate.  The  adults  of  this  species,  the 
S.  mansoni,  are  scarcely,  if  at  all,  to  be  distinguished  from  the  S. 
hamatobium.  Leiper  has  recently  noted  a  difference  in  that  the  male  of 


FIG.  67. — Anatomy  of  a  tape- worm,  T&nia  solium  (A.,  longitudinal,  B.,  cross 
section);  a  fluke,  Paragonimus  westermanii  (C).,  male  and  female  nematode,  Oxyuris 
vermicularis  (D.).  A.  i,  Testes;  2,  yolk  glands;  3,  shell  glands;  4,  ovary;  5,  vagina; 

6,  vas  deferens;  7,  uterus  before  branching;  8,  water-vascular  system.     B.  i,  Cuticle; 
2,  circular  muscle;  3,  ovary;  4,  testes;  5,  uterus;  6,  excretory  canal;  7,  nerve  cord. 
C.  i,  Oral  sucker;  2,  acetabulum;  3,  uterus;  4,  testes;  5,  excretory  canal;  6,  ovary; 

7,  yolk  glands.     D.  (a)  Female,     i,  Vulva;  2,  uterus;  3,  bulb  of  oesophagus;  4,  anus; 
(D)  Male.     i.  Bulbous  mouth  end;  2,  testes;  3,  spicule;  4,  alimentary  canal.     E. 
Egg.  of  P.  westermanii. 

S.  mansoni  has  seven  testicles  as  against  four  for  S.  hamatobium.  With 
S.  japonicum,  the  name  of  the  Eastern  species,  there  is  not  only  the 
difference  that  the  eggs  are  without  spines,  but,  in  addition,  the  skin 
of  the  adult  parasite  is  not  tuberculated,  as  is  the  case  with  the  other 
two  species.  It  is  slightly  smaller,  the  acetabulum  projects  more 
prominently,  and  the  lower  part  of  the  male  infolds  more  markedly 


302  FLAT    WORMS 

than  in  S.  hcematobium.  Catto  considers  that  the  S.  japonicum  may 
live  in  both  arteries  and  veins.  The  other  two  species  only  live  in 
branches  of  the  portal  vein.  The  blood  flukes  are  about  J£  inch  (13 
mm.)  long.  All  of  these  flukes  live  separately  until  maturity.  At 
this  time  the  female  enters  what  is  known  as  the  gynaecophoric  canal 
of  the  male;  this  canal  is  formed  by  the  infolding  of  the  sides  of  the  flat 
male  fluke,  thus  giving  a  rounded  appearance  to  the  male.  The 
female  is  longer  than  the  male  (about  %  inch  long),  and  is  thread-like 
and  of  a  darker  color.  Her  two  extremities  project  from  the  canal  of 
the  male  in  which  she  lives. 

The  oral  sucker  of  the  male  is  infundibuliform  and  is  smaller  than  the 
pedunculated  acetabulum.  In  the  female  the  oral  sucker  is  larger  than 
the  acetabulum.  The  eggs  of  S.  hcematobium  are  fusiform,  yellowish  in 
color,  have  a  thin  shell  and  a  terminal  spine. 

The  most  prominent  symptoms  of  the  Bilharz  disease  are  haemat- 
uria  and  bladder  irritation;  later  on  calculus  formation. 

In  rectal  bilharziasis  the  symptoms  are  more  those  of  bleeding  piles  or  of  a  mild 
dysentery. 

There  may  also  be  involvement  of  the  appendix. 

In  the  Japanese  infection  the  symptoms  point  more  to  liver  and 
spleen,  there  being  ascites,  cachexia,  and  a  bloody  diarrhoea.  Early 
in  the  infection  we  have  fever,  urticarial  spots  and  some  bronchial 
trouble  (urticarial  fever).  The  eggs  should  be  searched  for  in  the 
mucus  cap  on  the  faeces. 

The  eggs  of  the  S.  japonicum  are  readily  found  in  the  faeces;  they  are  about  100  X 
7o/i.  They  are  oval,  transparent,  and  with  a  smooth  shell,  within  which  can  be  made 
out  the  outlines  of  an  embryo.  Upon  adding  water  the  ciliated  embryo  begins  to 
show  movement  in  about  ten  minutes  and  shortly  afterward  bursts  out  of  the  shell 
and  swims  about  actively.  It  is  more  melon-shaped  than  the  miracidium  of  S. 
h&matobium. 

The  exact  life  history  is  not  known  of  any  of  these  flukes.  Looss  conjectures  that  it 
is  probable  that  the  miracidium  enters  the  skin,  not  requiring  an  intermediary  host. 
Frequent  experiments  have  failed  to  show  any  mollusk,  etc.,  which  attracted  the 
embryo.  Evidence  seems  to  show  that  those  who  are  constantly  wading  about  in  the 
water  of  the  pools  or  the  mud  of  the  fields  are  the  ones  most  subjected  to  infection. 

Katsurada,  by  experiments  with  a  cat  and  dog,  has  proved  that  infection  will  take 
place  through  the  shaved  skin  of  an  animal  held  in  infected  water — none  of  the  water 
being  allowed  to  enter  by  mouth.  Fully  developed  miracidia  and  the  male  and 
female  flukes  were  found  in  the  portal  vein.  It  is  thought  that  further  development 
of  the  miracidia  in  the  body  may  account  for  the  heavy  infection. 


LIFE  HISTORY  OF   SCHISTOSOMA 


303 


Recently  Leiper  has  found  cercariae  showing  the  absence  of  a  pharynx 
(characteristic  of  the  genus)  in  a  Japanese  mollusc.  Such  molluscs 
were  teased  out  in  water  and  laboratory  bred  mice  immersed  therein. 
One  of  these  mice  was  killed  a  month  later  and  adult  schistosomes 
were  found  in  the  portal  vessels.  Leiper  has  also  found  cercariae 
showing  absence  of  pharynx  in  four  different  species  of  molluscs  in 
Egypt.  With  such  molluscs  he  was  able  to  infect  white  rats  and 
other  animals.  He  states  that  infection  with  these  cercariee  from  the 
mollusc  host  can  bring  about  infection  either  by  way  of  the  mouth  or 
through  the  skin.  Sodium  bisulphate  in  a  strength  of  i  to  1000  killed 
these  cercariae  almost  immediately. 

It  would  therefore  seem  proven  that  all  human  schistosome  in- 
fections take  place  following  cercarial  and  not  miracidial  development. 
As  proof  that  S.  hcematobium  and  S.  mansoni  are  different  species, 
Leiper  notes  that  mice  infected  by  molluscs  of  the  genus  Bullinus 
showed  schistosomes  with  terminal  spined  eggs,  the  ovary  lying  in  the 
lower  half  of  the  female.  The  male  had  four  or  five  large  testes.  In 
mice  infected  by  molluscs  of  the  genus  Planorbis,  the  eggs  were  lateral 
spined,  the  ovary  was  in  the  anterior  half  of  the  body  and  the  male 

had  eight  small  testicles. 

• 

There  is  a  view  that  the  miracidium  enters  while  bathing  by  the  preputial  channel, 
hence  the  value  of  circumcision. 

If  urine  containing  eggs  is  diluted  with  water  the  miracidium  breaks  out  of  the  shell 
and  swims  about  as  if  in  search  of  some  desired  object. 

The  view  is  also  entertained  that  the  miracidium  may  gain  access  to  the  body 
through  the  drinking  water;  there  is  much  evidence  against  this.  However  access 
to  the  body  is  gained,  it  is  known  that  the  larval  forms  make  their  way  to  the  liver 
where  they  develop.  Arriving  at  maturity,  the  males  and  females  become  united 
and  proceed  to  the  terminal  branches  of  the  portal  vein,  where  the  irritating  eggs, 
given  off  by  the  female,  give  rise  to  the  symptoms. 

CESTODE  OR  TAPE-WORM  INFECTIONS 

The  cestodes  and  trematodes  constitute  the  two  great  divisions  of 
the  flat  worms.  Anatomically,  a  tape-worm  may  be  considered  as  a 
series  of  individual  flukes  united  in  one  ribbon-like  colony.  The 
cestode  segments,  or  proglottides  are  covered  by  an  elastic  cuticle  and 
in  their  interior  usually  contain  striated  elliptical  bodies  composed  of 
calcium  carbonate  about  5  to  25^  according  to  the  species  in  which  they 
are  found. 


3°4 


FLAT    WORMS 


These  calcareous  bodies  are  characteristic  of  cestode  tissue.  They  have  been 
mistaken  for  coccidia.  There  is  no  mouth  or  alimentary  canal  in  tape-worms,  the 
segments  absorbing  their  nourishment  through  the  general  surface. 

A  tape- worm  is  divided  into  the  segment-producing  controlling  head  and  the  series 
of  segments  or  proglottides  together  known  as  the  strobila.  The  head  and  neck 
together  form  the  scolex. 

The  head  contains  the  central  nervous  tissue  and  the  commencement 
of  the  water-vascular  system. 


DibothriocephaliA   latua 


Taenia   solium 


Taenia    saginata 


FIG.  68. — Adult  and  larval  stages  of  cestoda  of  man. 

Tape-worm  heads  are  provided  with  suctorial  or  hook-like  organs,  or  both,  to 
enable  them  to  hold  on  to  the  intestinal  mucosa. 

The  hooks  when  present  on  the  anterior  extremity  of  the  head  are  carried  by  a 
protrusible  structure  called  the  rostellum. 

The  importance  of  the  head  is  generally  recognized  by  the  well- 
known  fact  that  the  permanent  evacuation  of  one  of  these  parasites  is 
only  arrived  at  when  the  head  as  well  as  the  segments  is  expelled. 
Otherwise,  additional  segments  will  be  produced. 

Even  in  tape- worms  25  to  30  feet  in  length,  the  head  is  no  larger  than  a  small  shot. 
It  carries  the  suckers  or  booklets  which  best  enable  us  to  differentiate  the  different 


CE5TODE5  3°5 

species.  The  segments  adjacent  to  the  head  are  immature  —  the  sexually  mature 
ones  being  found  from  the  middle  of  the  body  onward. 

T.  sagifmia  has  about  2000  segments,  T.  solium  less  than  1000  whfle  T.  eckino- 
coccus  has  only  three  or  four.  The  sexually  mature  segment  possesses  a  varying 
number  ol  testicles:  three  in  Hymcnolepsis  nana  and  as  many  as  2000  in  Tcrnia 
sagittate.  As  with  the  flukes,  they  also  have  vasa  deferentia,  cirrus,  ovaries,  yolk 
glands,  uterus,  genital  pore,  etc.  The  location  of  the  genital  pore  and  the  charac- 
ter of  the  branching  of  the  uterus  are  of  the  greatest  importance  in  differentiation. 
The  sexually  mature  proglottides  may  either  expel  their  ova,  when  these  would  be 
found  in  the  faeces  or,  as  is  common,  they  break  off  and  pass  out  themselves  in  the 
faeces.  Then  they  either  expel  the  eggs  or  may  be  eaten  by  some  animal  and  in 
this  way  effect  an  entrance  for  their  ova.  It  is  an  important  practical  point  that 
the  faeces  of  a  patient  with  T.  solium  or  T.  sagittate  may  not  show  any  ova,  these 
passing  out  in  the  intact  segments.  The  oval  operculated  eggs  of  Diboikriocephalus 
lotus,  however,  are  constantly  in  the  faeces. 

The  "hexacanth"  or  six-hooked  embryo,  also  called  the  onchosphere,  is  the  essen- 
tial part  of  the  egg.  The  embryonic  envelope  is  dissolved  off  in  the  alimentary 
canal  of  the  animal  ingesting  it,  and  the  onchosphere  bores  its  way  through  the  gut 
to  later  become  encysted  in  various  tissues.  In  some  tape-  worms  a  ciliated  embryo 
is  liberated  from  the  egg  shell  and  swims  about  actively  to  enter  some  fish  or  other 
animal.  When  the  six-hooked  embryo  reaches  its  proper  tissue,  the  booklets  are 
discarded  and  a  scolex  similar  to  the  parent  one  is  developed.  At  this  time  we  have 
a  bladder-like  structure  with  the  scolex  inverted  in  it.  This  is  termed  the  proscolex 
stage.  This  Kttk  cyst  with  its  scolex  when  ingested  by  another  animal  is  digested, 
and  the  sccdfr,  establishing  itself  in  the  intestine,  develops  a  series  of  segments. 
The  ciliated  embryo  of  the  D.  lotus  does  not  form  a  cyst,  but  instead  a  worm-like 
creature  q'milar  to  the  adult.  This  is  termed  a  Plerocercoid. 


If  the  larval  stage  shows  a  single  cyst  and  a  single  head,  it  is  termed 
Cysticercus;  if  multiple  cysts  but  only  one  head  to  each  cyst,  Ccenwrus; 
while  with  multiple  cysts  and  multiple  heads  hi  each  cyst  the  term 
Eckinocvccus  is  used. 

Where  there  is  very  little  fluid  in  the  cyst  and  the  larva  is  of  minute 
as  with  the  HymenoUpsis,  the  term  Ccrcocystis  is  employed. 

KEY  TO  CESTODE  GENEEA 

I.  Head  with  two  elongated  sfit-Kke  suckers—  Genital  pores  ventral—  Rosette 
uterus.     Dibothriocephabdae. 

(A)  Single  set  of  genital  organs  in  each  segment.    Dtbotkrioccpkalus. 

(B)  Double  set  of  genital  organs  in  each  segment.    Dipbgottoporus. 

(O  Immature  forms  showing  characteristics  of  Dibothriocephalidae—  (collective 

group).     Sparganum. 
II.  Head  with  four  cup-like  suckers;  genital  pores  lateral     Taeniidae. 

(A)  Uterus  with  "M^Kaii  stem  and  a  varying  number  of  lateral  branches.     Tctnia. 

(B)  Uterus  without  median  stem  and  lateral  branches. 


FLAT   WORMS 

(1)  Genital  pores  single.     Rostellum  with  not  more  than  two  rows  of  hooks, 
(a)  Suckers  armed  with  numerous  small  booklets.     Fifteen  to  twenty 

testicles  in  each  segment.     Davainea. 

(6)  Suckers  not  armed.     Three  testicles  in  each  segment.     Hymeno- 
lepsis. 

(2)  Genital  pores   double.     Rostellum    with  four  or  five  rows  of  hooks. 

Dipylidium. 

TJENIID^E  INFECTIONS 

Tsenia  saginala  (Taenia  mediocanellata). — This  very  widely  dis- 
tributed tape-worm  is  often  termed  the  unarmed  tape-worm,  to 
distinguish  it  from  the  T.  solium  or  armed  tape- worm. 

It  is  from  10  to  25  feet  long  and  has  several  hundred  proglottides.  The  small 
pear-shaped  head  has  four  pigmented  elliptical  suckers  and  no  booklets.  The  seg- 
ments are  plumper  than  those  of  T.  solium,  hence  the  name  saginata.  The  single 
lateral  genital  pore  projects  markedly  and  in  a  series  of  segments  presents,  as  a  rule, 
first  on  one  side,  and  then  on  the  opposite  side  of  the  next  segment  (alternating). 
The  best  way  to  distinguish  a  segment  of  the  T.  saginata  from  the  T.  solium  is  by 
counting  the  number  of  lateral  uterine  branches;  these  number  fifteen  to  thirty,  are 
quite  delicate  and  branch  dichotomously.  The  lateral  divisions  of  the  uterus  of 
the  T.  solium  are  tree-like  in  their  branching  and  only  number  five  to  twelve  on  each 
side. 

T.  solium  has  three  ovaries  while  T.  saginata  has  only  two.  The  ox  is  the  inter- 
mediate host  of  T.  saginata.  The  eggs  of  Tcsnia  have  an  oval  outer  shell  which  is 
filled  with  rather  translucent,  refractile  yolk,  often  in  globules.  Within  the  oval 
shell  is  the  more  rounded  cell  of  the  six-hooked  embryo  with  its  thick  striated 
membrane.  The  outer  shell  is  often  absent  in  the  eggs  found  in  the  faeces,  only 
the  shell  of  the  six-hooked  embryo  being  found.  The  six-hooked  embryo,  having 
worked,  its  way  from  the  alimentary  canal  to  the  muscles  or  liver  of  the  ox, 
becomes  encysted  (Cysticercus  bovis).  This  little  bladder-like  structure  is  about 
Yi  by  y$  inch,  and  contains  but  a  small  amount  of  fluid. 

The  evaginated  head  does  not  show  booklets,  they  differing  from  the 
armed  rostellum  of  the  scolex  of  Cysticercus  cellulosce. 

Being  ingested  by  man's  eating  raw  or  imperfectly  cooked  meat,  the  adult  stage 
becomes  established  in  his  alimentary  canal  in  about  two  months. 

It  is  probable  that  the  various  raw-meat  cures  have  made  the  infection  more  com- 
mon. Cysticercus  bovis  is  more  abundant  in  the  tongue  of  cattle  than  elsewhere  in 
the  musculature. 

For  this  reason  it  would  seem  advisable  to  use  other  raw  meat  than  beef  in  such 
cures. 

In  Abyssinia  the  infection  is  said  to  be  universal,  and  a  man  without  a  tape- 
worm to  be  a  freak.  An  important  point  is  the  fact  that  the  larval  stage  almost 
never  appears  in  man.  It  is  this  fact  which  makes  it  a  so  much  less  dangerous  para- 
site than  the  T.  solium,  which  readily  establishes  a  larval  existence  in  man  if  the  ova 


HYMENOLEPIS  307 

are  introduced  into  the  human  stomach.  Cooking  meat  always  destroys  the 
cysticercus.  A  period  of  about  two  months  elapses  after  the  ingestion  of  the  cysti- 
cercus  before  the  mature  segments  pass  out  of  the  rectum.  These  not  only  make 
their  exit  with  the  faeces,  but  are  also  capable  of  wandering  out  at  other  times.  In 
this  they  differ  from  the  segments  of  T.  solium.  T.  saginata  next  to  Hymenolepis 
nana  is  the  common  tape-worm  of  the  United  States.  Dr.  Stiles  has  examined 
several  hundred  tape-worms  in  the  United  States  during  the  past  few  years  and 
has  found  only  one  T.  solium. 

In  Paris  Blanchard  found  1000  T.  saginata  to  21  T.  solium.  Certain 
German  statistics,  however,  show  about  one-half  as  many  T.  solium 
as  T.  saginata. 

Abnormalities  of  the  scolex  and  proglottides  are  not  uncommon  with  T.  saginata. 
This  is  less  frequently  the  case  with  T.  solium. 

Taenia  solium. — The  measly  pork  tape-worm  is  smaller  than  the 
T.  saginata  and  differs  from  it  in  having  a  globular  head,  with  a  ros- 
tellum  which  is  crowned  by  26  to  28  booklets. 

In  T.  saginata  a  depression  takes  the  place  of  the  armed  rostellum;  the  suckers 
of  T.  saginata  are,  however,  much  more  powerful  than  those  of  T.  solium.  The 
segments  have  only  five  to  ten  coarse  branches  and  are  expelled  only  at  the  time  of 
defecation.  The  segments  or  the  ova  having  been  ingested  by  a  hog,  the  six-hooked 
embryo  is  liberated  and  becomes  encysted  chiefly  in  the  tongue,  neck,  and  shoulder 
muscles  of  the  hog,  as  an  invaginated  scolex.  Pork  containing  this  cysticercus 
(Cysticercus  celluloses)  is  known  as  measly  pork.  This  cysticercus  contains  much 
more  fluid  than  that  of  the  ox  and  is  from  %  to  ^  inch  long.  If  one  by 
chance  should  carry  the  egg  on  his  fingers  to  his  mouth,  as  the  result  of  examining 
mature  segments,  the  larval  stage  may  be  established  in  man.  If  this  infection  is 
not  heavy,  very  few  symptoms  may  be  observed.  The  cysticercus,  however,  tends 
to  invade  the  brain,  next  in  frequency  the  eye,  and  so  causes  convulsions,  death  or 
blindness.  Instead  of  only  being  the  size  of  a  pea,  these  cysts,  when  forming  in  the 
brain,  may  be  the  size  of  a  walnut  or  larger.  T.  solium  is  comparatively  common  in 
north  Germany,  but  is  exceedingly  rare  in  England  and  the  United  States. 

Taenia  africana. — This  is  an  unarmed  tape-worm,  only  about  5  feet  long.  It 
was  found  in  a  native  soldier  in  German  East  Africa. 

Garrison  has  reported  from  the  Philippines  a  tape-worm  with  an  unarmed 
rostellum,  V-shape  and  spiral  formation  of  the  uterine  stem  with  compact  structure 
of  the  gravid  uterus  under  the  name  of  Tania  philippina.  Another  tape-worm, 
T.  confusa  of  which  only  segments  were  found  was  reported  by  Ward  from  Nebraska. 

Hymenolepis  nana  (Tsenia  nana). — This  is  generally  known  as  the 
dwarf  tape- worm — it  is  the  smallest  of  the  human  tape-worms.  It  is 
from  Y±  inch  to  ^  inch  in  length,  and  is  less  than  >^5  inch  in  breadth. 
(10  X  i  mm.) 


308 


FLAT   WORMS 


The  genus  Hymenolepis  has  lateral  genital  pores,  all  of  which  are  on  the  same 
side.  These  lateral  genital  pores  cannot  be  made  out  in  specimens  as  ordinarily 
examined.  The  head  has  four  suckers  and  a  rostellum,  which  is  usually  invaginated. 
The  rostellum  has  a  single  row  of  24  to  30  booklets  encircling  it.  Of  the  150  to 
200  narrow  segments  the  terminal  ones  are  packed  with  eggs  which  in  the  last  two 
or  three  seem  to  fill  entirely  the  disintegrating  segments.  It  would  seem  that  the 
fully  mature  segments  disintegrate  and  in  this  way  the  eggs  are  set  free  in  the 
surrounding  intestinal  contents. 

The  worms  as  found  in  fresh  faeces  after  taeniacide  treatment  are  frequently  in  an 


a- Laminated  elastic   cuticle 
b  •  fmer  yermmal    layer 
c  -  Scoitr  frte  In  cyst 
d- Brood   capsule 

;-  Endogenous  daughter  cyst 
-  RufJurvd    brood  capsule 


-b. 


HYMENOLEPIS 
DIMINUTA. 


FIG.  69. — Other  cestodes  of  man. 

advanced  state  of  disintegration  so  that  it  is  impossible  to  make  out  the  head  or 
booklets. 

The  eggs  of  this  species  are  quite  characteristic,  there  being  two  distinct  mem- 
branes. The  inner  one  has  two  distinct  knobs,  from  which  thread-like  filaments 
proceed.  The  eggs  of  the  H.  diminuta  have  a  thicker,  striated,  outer  membrane 
and  there  are  no  filaments.  The  eggs  of  the  Dipylidium  caninum  are  similar,  but  are 
found  in  the  faeces  in  aggregations — several  eggs  in  a  packet. 

The  dwarf  tape-worm  has  been  found  to  be  the  most  common  tape- 
worm in  the  United  States.  Dr.  Stiles  found  it  in  about  5%  of  children 
in  a  Washington  orphanage. 


DIBOTHRIOCEPHALUS 


309 


It  has  been  estimated  that  in  certain  parts  of  Italy  10%  of  the  children  may  be 
infected.  The  symptoms,  especially  nervous  ones,  may  be  marked  in  this  infection. 
It  has  been  incriminated  as  a  cause  of  chyluria.  Although  very  small,  yet  the  num- 
ber of  parasites  may  be  very  great,  even  more  than  1000.  In  a  case  that  I  treated 
with  thymol  there  were  1500  worms  expelled.  A  form  found  in  rats,  which  may  be 
identical  with  H.  nana,  does  not  require  an  intermediate  host.  The  six-hooked 
embryo  bores  into  the  intestinal  villus  and  there  develops  a  Cercocystis  (larva  of 
small  dimensions  with  but  little  fluid).  When  fully  developed,  it  drops  into  the 
lumen  of  the  gut,  and  a  new  parasite  is  added  to  the  already  existing  number  of 
parasites.  This  explains  the  heavy  infection.  H.  diminuta  and  H.  lanceolata  have 
also  been  reported  for  man  a  few  times. 

H.  diminuta  is  much  larger  than  H.  nana,  being  about  10  inches  long.  The 
suckers  are  small  and  the  rostellum  insignificant  and  unarmed.  The  intermediate 
host  in  some  insect,  as  a  moth;  the  definitive,  the  rat.  As  man  is  not  liable  to  eat 
the  insect  hosts  the  infection  is  rare  in  man.  Twelve  cases  have  been  reported  for 
man  of  which  five  were  from  the  U.  S. 

H.  lanceolata  is  common  in  geese  and  ducks. 

Dipylidium  canimim  (Taenia  cucumerina)  (T.  flavopunctata). — This  is  a  common 
parasite  of  dogs  and  cats.  The  larval  stage  is  passed  in  lice  and  fleas.  The  cases  of 
human  infection  have  been  principally  in  children,  probably  from  getting  dog  lice 
or  fleas  in  their  mouths.  The  number  of  infections  reported  for  man  is  about  40 
and  of  these  about  30  in  children.  The  head  has  four  suckers  and  a  rostellum, 
which  has  three  or  four  rows  of  encircling  booklets.  The  segments  have  the  shape 
of  melon  seeds  and  have  bilateral  genital  pores. 

Davainea  madagascariensis. — This  tape-worm  has  been  found  in  Siam  and 
Mauritius.  It  is  about  10  inches  long.  The  head  has  four  suckers  and  a  rostellum 
with  90  booklets.  The  suckers  have  rings  of  booklets.  The  genital  pores  are 
unilateral.  The  cockroach  is  supposed  to  be  the  intermediate  host. 

There  have  been  about  10  cases  reported  (Madagascar,  Siam  and  British  Guiana). 
There  has  also  been  reported  a  D.  asiatica,  the  single  specimen,  however,  lacking  a 
head  so  that  the  exact  genus  is  doubtful.  It  has  been  reported  twice  in  children  in 
Breslau.  The  intermediate  host  is  thought  to  be  a  cyclops.  Garrison  reported 
cases  from  the  Philippines. 

DlBOTHRIOCEPHALID^E  INFECTIONS 

Dibothriocephalus  latus  (Bothriocephalus  latus). — This  is  fre- 
quently termed  the  broad  Russian  tape-worm.  It  has  a  small  olive- 
shaped  head  with  two  deep  winding  suctorial  grooves  on  each  side;  it 
has  neither  rostellum  nor  hooklets. 

The  segments  are  quite  broad,  being  about  /^  by  3^  inch.  At  the  end  of  the 
strobila  they  are  more  nearly  square.  The  segments  are  very  numerous,  3000  or 
more.  The  fully  developed  worm  is  about  30  feet  long.  The  uterus  in  each  segment 
is  rosette-shaped  and  the  genital  pore  is  ventrally  situated.  The  eggs  of  this  species 
have  an  operculum  and  a  ciliated  embryo.  This  ciliated  embryo  swims  around  and 
either  enters  some  fish,  especially  pike,  directly  or  through  an  as  yet  unknown  inter- 


3io 


FLAT    WORMS 


mediary.  This  parasite  produces  an  intense  anaemia  similar  to  pernicious  anaemia. 
It  is  a  frequent  parasite  in  Switzerland,  Bavaria,  Japan,  Scandinavia,  and  Russia. 
Recently  several  cases  have  been  reported  from  our  Northwest,  and  some  of  the  fish 
of  the  waters  of  that  region  are  said  to  be  infected.  The  larva  is  a  pleroceroid  and 
is  about  i  inch  long.  It  is  said  that  salting,  smoking,  or  other  ordinary  methods  of 
preserving  fish  will  not  kill  it. 

A  tape-worm,  Diplogonoporus  grandis  has  been  reported  from  Japan.     In  this 
there  are  two  complete  sets  of  genital  organs  to  each  segment. 


SOMATIC  T^NIASIS 

While  rarely  we  may  have  the  larval  stage  of  T.  solium  present  in 
man,  and  while  certain  bothriocephalid  larvae  (Sparganum  mansoni 
and  Sparganum  proliferum)  infect  man,  yet  they  are  unimportant  as 
compared  with  the  larval  stage  of  the  Tcenia  echinococcus. 


FIG.  70, — Daughter  cyst  from  hydatid 
cyst,  considerably  enlarged.     (Coplin.) 


FIG.  71. — A  group  of  daughter  cysts 
from  hydatid  cysts.     (Coplin.) 


Taenia  echinococcus. — The  adult  stage  of  this  parasite  is  passed  in 
dogs.  It  is  one  of  the  smallest  tape-worms  known,  being  only  about 
3^5  inch  long.  It  has  a  head  with  four  suckers  and  a  rostellum  en- 
circled with  hooks.  There  are  only  three  to  four  segments.  The  larval 
stage,  on  the  contrary,  gives  one  of  the  largest  of  larval  cestodes.  In 
man  it  may  reach  the  size  of  a  child's  head. 

The  larval  stage  is  also  found  in  hogs  and  sheep,  and  it  is  probable  that  by  reason 
of  the  dog's  eating  the  echinococcus  cyst  of  such  animals  at  the  abattoir  we  owe 
the  increase  in  this  serious  infection. 

Man  contracts  the  infection  from  association  with  dogs.  The  disease  is  peculiarly 
prevalent  in  Iceland.  As  stated  above,  the  adult  stage  is  passed  in  the  intestine  of 


HYDATIDS 


the  dog.  Should  the  egg-bearing  segments  passed  by  the  dog  contaminate  the  hands 
of  man  and  a  single  egg  be  ingested,  we  may  have  hundreds  of  Tania  larvae  produced. 
The  six-hooked  embryo,  leaving  its  shell,  bores  its  way  through  the  walls  of  the 
alimentary  tract  and  especially  seeks  the  liver,  just  as  the  embryo  of  T.  solium  seeks 
the  brain  and  eye. 

Griffith  notes  that  in  Australia  from  10  to  15%  of  hydatid  cysts 
occur  in  the  lungs.  The  cyst  wall  is  quite  thin  and  the  hydatid 
cachexia  seems  to  appear  earlier  in  the  lung  than  in  the  liver  cases. 


Ova  or  the  Parasitic  Worms  or  Man 
CESTODA 

ORAWM  TO  SCALE  X  IGOO 


i^iuiu-  ^*=^-    Dibothrio-  —  - — - 

gonoporus    cephalus       nymenojepisimna 

Hymenolepi 

^  diminuta 


Toenia^Bf 

vta^  \^,^'r 

Dipylidium 

Cestode  segments 

DRAWN     TO    SCALE    X    IO        O 

Da\5in  /     U^l 

m^^?^SFiensis  H^no-    / 

lepis         /«    l**?^* 


FIG.  72. — Cestode  ova. 

In  the  development  of  the  cyst,  after  the  embryo  has  come  to  rest  at  some  point 
in  the  liver,  we  have  formed  at  first  an  indistinctly  laminated  external  envelope 
with  coarsely  granular  fluid  contents.  Later  on  the  contents  become  transparent, 
and  two  distinct  layers  can  be  observed:  i.  The  external,  markedly  laminated  one, 
and  2.  the  internal  one,  made  up  of  small  cells  externally  and  large  cells  and  cal- 
careous corpuscles  internally.  This  internal  lining  membrane  is  known  as  the  paren- 
chymatous  or  germinal  layer.  When  the  external  layer  is  incised  it  curls  up  by 
reason  of  its  elasticity.  This  is  characteristic  of  such  a  cyst.  In  addition,  we  have 
an  enveloping  connective-tissue  capsule  formed  by  the  surrounding  liver  substance. 


312  FLAT   WORMS 

From  the  germinal  layer  arise  the  brood  capsules  and  the  scolices.  In  these  brood 
capsules  we  have  the  cellular  layer  external — just  the  reverse  of  the  mother  cyst. 
Scolices  may  develop  either  on  the  outside  or  inside  of  these  brood  capsules.  It  is 
interesting  to  note  that  one  onchosphere  may  develop  hundreds  of  scolices.  From 
the  parenchymatous  layer  of  the  mother  cyst,  daughter  cysts  are  formed;  these  have 
an  external  stratified  layer  and  an  internal  parenchymatous  one;  within  them  a  vary- 
ing number  of  scolices  may  develop.  From  these  daughter  cysts,  granddaughter 
cysts  may  arise — all  within  the  mother  cyst — and  hence  are  termed  endogenous. 

At  times  the  daughter  cysts  work  their  way  external  to  the  mother  cyst  and  pro- 
ceed to  develop  in  a  manner  similar  to  the  endogenous  formation.  The  exogenous 
development  is  rare  in  man,  but  common  in  hogs.  Hydatids  containing  no  scolices 
are  called  sterile.  These  cysts  may  be  as  large  as  a  child's  head,  but  are  usually 
smaller.  The  fluid  of  these  cysts  contains  about  i%  of  NaCl,  also  a  trace  of  sugar; 
in  addition  there  is  a  toxin  which  produces  urticaria  and  acts  as  a  cardiac  depressant. 
If  any  quantity  should  escape  into  the  peritoneal  cavity  at  operation,  it  may  cause 
death.  Hydatids  develop  very  slowly,  and  the  duration  of  the  disease  is  usually 
from  two  to  eight  years. 

Echinococcus  multilocularis  is  possibly  due  to  a  species  different  from  T.  echino- 
coccus.  In  this  we  have  a  honeycomb  arrangement  with  cavities  filled  with  a  gela- 
tinous material.  The  majority  of  these  cysts  are  without  scolices.  This  form  of 
hydatid  is  very  fatal. 

Sparganum  mansoni  (Bothriocephalus  liguloides). — This  is  a  larval  bothrio- 
cephalid  which  is  about  5  to  10  inches  long  and  has  been  reported  10  times  in  Japan. 
It  has  been  found  in  various  parts  of  the  body,  as  in  pleural  cavity,  tissues  about 
kidney,  and  in  abscess  of  the  thigh.  They  have  been  found  in  the  urethra  and  under 
the  conjunctiva.  They  resemble  ribbon-like  strings  of  fat. 

Sparganum  prolifer  (Plerocercoides  prolifer).— This  has  been  reported  from 
Japan  as  a  larval  form  in  the  subcutaneous  tissue.  Stiles  has  found  these  larval 
forms  in  skin  lesions  in  Florida.  They  show  themselves  as  bizarre  grub-like  forms. 
They  reproduce  by  budding. 


CHAPTER  XVIII 
THE  ROUND  WORMS 

CLASSIFICATION  OF  THE  NEMATHELMINTHES  (ROUND  WORMS) 


Class 


Nematoda 


Family 
Angiostomidae 


Filariidae 


Trichotrachelidae 


Strongylidae 


Ascaridae 


Genus 

Species 

Strongyloides 

S.  stercoralis. 

Dracunculus 

D.  medinensis 

F.  bancrofti 

F.  loa 

F.  perstans 

Filaria 

F.  demarquayi 

F.  ozzardi 

F.  philippinensis 

Onchocerca 

O.  volvulus 

f  Trichuris 

T.  trichiura 

1  Trichinella 

T.  spiralis 

Eustrongylus 

E.  gigas 

Strongylus 

S.  apri 

Trichostrongylus 

T.  instabilis 

Triodontophorus 

T.  deminutus 

(Esophagostoma 

O.  brumpti 

Physaloptera 

P.  caucasica 

Ancylostoma 

A.  duodenale 

Necator 

N.  americanus 

Ascaris 

A.  lumbricoides 

Belascaris 

B.  mystax 

Oxyuris 

O.  vermicularis 

Gigantorhynchus 

G.  gigas 

'  Hirudo 

H.  medicinalis 

Limnatis 

L.  nilotica 

Haemadipsa 

H.  ceylonica 

Acanthocephali 


Hirudinei 


NOTE. — The  Strongyloides  stercoralis  was  formerly  described  under  two  desig- 
nations: (i)  Anguillula  inteslinalis,  a  parasitic  generation  and  (2)  Anguillula  ster- 
coralis, a  free  living  generation. 


ROUND  WORMS  OR  NEMATODES 

All  nematodes  are  covered  by  a  cuticle  which  varies  in  thickness, 
and  is  frequently  ringed.    The  cuticle  is  moulted  three  or  four  times. 

313 


THE   ROUND   WORMS 


rule. 


The  cuticle  is  formed  by  the  underlying  ectoderm  which  is,  as  a  rule, 
markedly  developed  in  four  ridges  which  divide  the  body  into  quad- 
rants. Within  the  ectoderm  is  the  body  cavity,  a  space  in  which  the 
reproductive  organs  lie  in  a  clear  fluid.  The  excretory  system  usually 
consists  of  two  tubes  which  discharge  near  the  head. 

While  the  alimentary  canal  is  more  or  less  tube  like  in  appearance  it  shows  near 
the  mouth  a  muscular  oesophagus  with  a  bulb-like  expansion  at  the  commencement 
of  the  remainder  of  the  intestinal  tract.  There  is  a  nerve  ring  around  the  oesophagus. 

The  testis  and  ovary  are  generally  tube  like.  The  sexes  are,  as  a  rule,  separate. 
The  male  can  usually  be  recognized  by  its  smaller  size,  its  curved  or  curled  posterior 
end,  and  at  times  exhibiting  an  umbrella-like  expansion — the  copulatory  bursa. 
The  spicules,  chitinous  copulatory  structures,  may  be  observed  drawn  up  in  the  worm 
or  projected  out  of  the  cloaca.  The  genital  opening  of  the  female  is  ventral  and 
usually  about  the  mid-point;  that  of  the  male  is  close  to  the  anus. 

Certain  papillae  in  the  region  of  the  anus  are  valuable  in  differentiation.  As  a 
rule,  nematodes  develop  in  damp  earth  from  the  eggs  as  rhabditiform  larvae.  Very 
few  nematodes  are  viviparous  (Filaria,  Trichinella)  being  usually  oviparous  (As- 
caris)  less  frequently  ovo viviparous  (Oxyuris). 

The  families  Gnathostomidae  and  Anguillulidae  are  of  very  little 
importance  in  human  parasitology.  Gnathostoma  siamense  was  once 
found  in  a  breast  tumor  and  Rhabditis  pellio  once  in  the  urine. 

Anguillula  aceti,  the  vinegar  eel,  has  been  reported  from  the  genito-urinary  tract 
several  times.  Such  cases  can  be  explained  by  the  prior  contamination  of  the  urine 
bottle  or  by  the  use  on  the  part  of  the  patient  of  a  vinegar  vaginal  douche.  The 
genera  Rhabditis  and  Anguillula  belong  to  the  family  Anguillulidae. 

A  case  of  infection  with  a  small  nematode  found  in  the  papules  of  a  skin  infection, 
in  a  French  boy  is  recorded  as  due  to  Rhabditis  niellyi.  The  present  view  is  that 
the  parasites  were  embryos  of  A.  duodenale,  boring  into  the  skin. 

ANGIOSTOMID,E 

In  this  family  we  have  heterogenesis. 

Strongyloides  stercoralis. — This  parasite  was  formerly  thought 
to  be  the  cause  of  Cochin-China  diarrhoea.  It  presents  two  genera- 
tions: i.  Parasitical  or  intestinal  form.  2.  The  free  living  or  faecal 
form. 

i.  The  intestinal  form  (also  known  as  Anguillula  intestinaHs}  is  represented  only 
by  females.  These  are  about  ^2  inch  (2  mm.)  long  and  reproduce  partheno- 
genetically.  They  have  a  pointed,  four-lipped  mouth,  and  a  filariform  oesophagus 
which  extends  along  the  anterior  fourth  of  the  body.  The  anus  is  situated  near  the 
sharpened  posterior  end,  the  vulva  about  the  lower  third  of  the  body.  The  uterus 
contains  a  row  of  eight  to  10  elliptical  eggs  which  stand  out  prominently  in  the  pos- 


THE  GUINEA  WORM  315 

terior  part  of  the  body  by  reason  of  being  almost  as  wide  as  the  parent  worm.  They 
usually  live  deep  in  the  mucosa  and  the  embryos  emerge  from  the  ova  laid  in  the 
mucosa.  The  embryos  escape  from  the  eggs  while  still  in  the  intestine,  so  that  in 
the  faeces  we  only  find  actively  motile  embryos.  The  eggs,  which  are  strung  out 
in  a  chain,  never  appear  in  the  fasces  except  during  purgation.  As  they  greatly 
resemble  hookworm  eggs,  this  is  a  point  of  great  practical  importance.  In  fresh 
faeces  we  find  hookworm  eggs  and  Strongyloides  embryos.  The  embryos  are  rather 
common  in  stools  in  the  tropics.  These  embryos  have  pointed  tails  and  are  about 
250  X  i3M-  They  have  a  double  cesophageal  bulb.  They  are  about  250*1  when  they 
first  emerge  but  may  grow  until  they  will  approximate  500/1  in  the  fseces.  If  the 
temperature  is  low,  these  rhabditiform  embryos  develop  into  filariform  embryos, 
which  being  ingested  form  the  infecting  stage.  It  has  been  demonstrated  that 
infection  of  man  may  also  take  place  through  the  skin.  If  the  temperature  is  warm, 
25°  to  35°C.,  these  embryos  develop  into: 

2.  The  free  living  form,  Anguillula  stercoralis.  In  this  we  have  males  and  females, 
with  double  cesophageal  bulbs,  the  male  about  ^0  inch  (%  mm.)  long  with  an  in- 
curved tail  and  two  spicules  and  the  female  about  %5  inch  (i  mm.)  long  with  an 
attenuated  tail;  these  copulate  and  we  have  produced  rhabditiform  larvae,  which 
later  change  to  filariform  ones.  At  this  time  the  length  is  about  550  microns. 
These,  being  ingested,  start  up  the  parasitical  generation.  If  these  do  not  reach  the 
intestine  they  die  out. 

FILARIID.E 

This  family  is  of  the  greatest  importance  to  man.  It  is  also  one 
about  which  much  confusion  exists  as  to  the  adult  type;  hence  anyone 
finding  adult  filariae  should  fix  them  in  hot  5%  glycerine  alcohol 
(alcohol  70%),  and  subsequently  mount  in  glycerine  gelatin.  Formalin 
is  not  to  be  used,  other  than  for  a  very  brief  period  (two  to  six  hours) 
and  then  followed  by  the  lacto-phenol  method. 

These  worms  are  most  likely  to  be  seen  as  writhing  thread-like  worms,  especially 
in  the  lymphatic  glands  and  connective  tissue,  and  about  body  cavities.  They  have 
a  lipped  or  simple  mouth  and  a  filariform  oesophagus.  The  male  has  an  incurved 
tail  with  preanal  and  postanal  papillae  which  may  be  even  corkscrew-like  as  in  F. 
immitis.  The  spicules  are  unequal  or  there  may  be  but  one.  The  female  is  ovovivi- 
parous,  the  vulva  is  at  the  anterior  end  and  the  uterus  usually  double. 

Dracunculus  medinensis  (Filaria  medinensis). — The  Guinea  or 
Medina  worm,  of  which  until  recently  only  the  female  was  known,  is 
of  great  importance  in  parts  of  India,  Africa,  and  Arabia.  The  female 
is  a  thread-like  worm,  about  20  to  30  inches  long.  The  habitat  is  the 
subcutaneous  and  intermuscular  connective  tissue,  especially  of  the 
lower  extremity.  It  develops  without  symptoms.  Finally  a  blister- 
like  area  appears  on  the  surface  of  the  leg,  particularly  about  ankle- 
joint,  which  soon  forms  a  painful  ulcer.  From  this  opening  the 


THE    ROUND    WORMS 


ryos 


anterior  end  of  the  worm  projects  to  pour  forth  its  striated  embry 
upon  contact  with  water. 

The  mouth  is  terminal  and  the  body  uniformly  cylindrical.     The  uterus  is  a  con- 
tinuous tube  filled  with  sharp-tailed,  transversely  striated  embryos,  650  X  i7/i,  and 


Fig.  73. — (la)  Adult  female  Guinea  worm  (Dracuncidus  medinensis}  showing 
anchoring  hook  at  posterior  extremity,  (ib)  Cross  section  of  female  Dracunculus 
showing  uterus  filled  with  embryos,  (ic)  Striated  embryos  of  the  Guinea  worm, 
(id)  Cyclops  coronatus,  the  minute  crustacean  which  serves  as  the  intermediate  host 
of  D.  medinensis.  (2a-2d)  Anterior  and  posterior  extremities  of  F.  loa.  (zc)  Sec- 
tion showing  tuberculated  cuticle.  (2b),  Male  and  female  F.  loa,  natural  size.  (3a) 
Bulbous  anterior  extremity,  Filaria  bancroft.  (36)  Tail  of  male.  (3c)  Tail  of  female. 
(3d).  Male  and  female,  natural  size  of  F.  bancrofti.  (4a)  Tumor  mass  of  F.  volvulus 
laid  open.  5,  Mosquito  showing  filarial  embryos  in  thoracic  muscles  (a)  and  in 
labium  (b).  The  labella  which  are  separated  from  the  labium  by  Button's  mem- 
brane are  seen  at  (c).  6.  (a)  Embryo  of  F.  bancrofti  (b)  embryo  of  F.  loa  showing 
filling  of  tail  end  with  cells.  7,  Micrafilaria  of  F.  bancrofti  in  blood.  Dotted  lines 
show  location  in  break  in  cells  column  and  V  spot. 


constitutes  the  greater  part  of  the  body,  the  alimentary  canal  being  pressed  to  one 
side.  The  genital  organs  probably  discharge  through  the  oesophagus.  The  body 
when  being  extracted  is  rather  transparent.  The  tip  of  the  tail  is  bent,  forming  a 
sort  of  anchoring  hook.  Recently  Leiper  fed  monkeys  on  bananas  containing  in- 
fected Cyclops,  and  at  the  autopsy  six  months  later  obtained  both  male  and  fe- 
male forms. 


FILARIAL  WORMS  317 

As  regards  the  life  history,  Fedschenko,  in  1870,  showed  that  the  embryos  when 
liberated  swam  around  in  water  and  finally  entered  the  bodies  of  species  of  the  genus 
Cyclops.  The  female  tends  to  come  to  the  surface  in  the  lower  extremities,  and 
experiments  show  that  if  on  the  blister-like  points  of  emergence  some  water  be 
squeezed  out  from  a  sponge,  the  uterus  will  eject  a  milky-looking  fluid  containing 
myriads  of  embryos.  This  would  indicate  that  the  worm  selects  the  lower  extremity 
so  that  the  embryos  may  gain  access  to  the  Cyclops  when  the  host  is  wading  through 
the  water. 

Leiper  showed  that  a  strength  of  HC1  equal  to  that  of  gastric  juice  killed  the 
Cyclops,  but  made  the  Dracunculus  embryos  very  active.  From  this  he  judged  that 
infection  must  probably  take  place  from  drinking  water  containing  infected  Cyclops. 
The  suggestion  of  Leiper  that  wells  harboring  Cyclops  be  treated  with  steam,  intro- 
duced by  a  pipe,  seems  to  be  valuable.  The  disease  is  known  as  "Dracontiasis." 

Filaria  loa  (Filaria  oculi). — This  is  a  thread-like  worm  of  west 
Africa  about  i  to  2  inches  long.  The  cuticle  is  characterized  by 
distinct  wart-like  structures. 

The  anterior  extremity  is  like  a  truncated  cone  with  two  papillae  at  the  base  of 
the  cone.  The  wart-like  cuticular  protuberances  or  bosses  are  about  12  to  15 
microns  in  height.  The  females  are  2  to  3  inches  (50  to  70  mm.)  long  and  about  % 
mm.  broad. 

The  males  are  smaller  than  the  females  and  have  three  preanal  papillae  and  two 
postanal  ones.  There  are  two  short  unequal  spicules.  The  life  history  is  not  satis- 
factorily established.  The  young  are  born  ovoviviparously,  and  it  has  been  sug- 
gested that  the  localized  cedemas,  known  as  Calabar  swelling,  may  be  due  to  the 
irritation  produced  by  these  eggs.  These  swellings  are  of  hen's  egg  size,  painless, 
do  not  pit  on  pressure  and  last  about  three  days.  They  occur  especially  on  the  hands 
and  arms.  The  embryos  almost  exactly  resemble  those  of  F.  bancrofti.  They  have 
a  diurnal  periodicity,  however,  appearing  in  the  blood  about  8  A.  M.,  increasing  to 
noon  and  disappearing  about  9  p.  M.  The  adult  worms  have  a  tendency  to  wander 
about  in  the  subcutaneous  connective  tissue,  especially  about  the  region  of  the  orbit 
or  even  under  the  conjunctiva. 

Adult  worms  of  F.  loa  have  been  found  and  extracted,  with  an  absence  of  the 
filarial  embryos  in  the  peripheral  circulation  of  the  patient.  While  immature  adult 
worms  have  been  extracted  from  children  the  embryos  have  only  exceptionally  been 
found  in  these  children.  This  would  speak  for  a  very  long  developmental  period 
for  the  adult  worm  and  as  a  matter  of  fact  the  infection  often  only  shows  itself  years 
after  the  opportunity  for  infection. 

Leiper  has  just  noted  two  species  of  Chrysops  (Mangrove  Flies)  as  intermediate 
hosts,  the  embryos  developing  in  the  salivary  glands. 

Filaria  bancrofti  (Filaria  sanguinis  hominis). — This  is  the  most 
important  of  the  filarial  worms.  It  is  a  common  infection  in  south 
China,  India,  the  West  Indies,  and  in  the  Pacific  Islands,  especially 
Samoa. 


318  THE   ROUND   WORMS 


f  -I -M  -I  C 


In  medical  books  the  embryos  have  been  designated  Filaria  sanguinis  hominis. 
This  species  is  the  cause  of  the  common  manifestations  of  filariasis,  such  as  elephanti- 
asis, varicose  groin  glands,  chyluria,  lymph  scrotum,  etc. 

Filarial  diseases  are  prone  to  lymphangitis  attacks.  Thus  in  lymph  scrotum  an 
erysipelatoid  condition  of  the  scrotum  with  high  fever  and  chills  may  result.  This 
condition  is  at  times  mistaken  for  malaria.  Varicose  groin  glands  may  be  mistaken 
for  hernia.  In  the  Philippines  very  few  symptoms  are  noted  in  those  affected  with 
filariasis.  Occasionally  chylocele  or  chyluria  is  reported. 

F.  bancrofti  lives  in  lymphatics  of  trunk  and  extremities.  At  times  the  fine  white 
thread-like  worms  may  be  seen  as  writhing  coils  in  lymphatic  glands. 

The  sexes  are  usually  found  together.  The  females  are  about  3  inches  long 
and  the  males  less  than  2  inches.  The  tails  of  both  sexes  are  incurved,  but  that  of 
the  male  is  more  so.  The  head  is  club-shaped.  The  vulva  opens  1.2  mm.  from  the 
anterior  end.  There  are  two  uterine  tubules.  The  sheathed  embryos  are  supposed 
to  be  born  viviparously  and  Manson  supposes  that  as  a  result  of  injury  to  the 
parent  worm  and  resulting  extrusion  of  eggs,  the  blocking  of  lymph  channels  occurs. 

A  very  interesting  fact  is  that  people  with  elephantiasis  fail  to  show  larvae  in  the 
peripheral  circulation.  Manson  considers  that  it  is  due  to  the  blocking  of  the 
lymph  channels. 

These  embryos  show  a  nocturnal  periodicity.  During  the  day  they 
remain  in  the  lungs,  and  larger  arteries. 

If  the  patient  sleeps  in  the  daytime  and  is  active  at  night  the  nocturnal  perio- 
dicity or  presence  of  embryos  in  peripheral  circulation  is  inverted.  In  the  case  of 
F.  loa,  however,  a  change  of  habits  does  not  change  the  periodicity  of  the  filarial 
embryos,  they  continue  to  appear  in  the  peripheral  circulation  by  day  even  if  the 
patient  sleeps  at  that  time. 

The  disease  is  transmitted  especially  by  Culex  fatigans.  The  sheathed  embryos, 
getting  into  stomach  of  mosquito,  wriggle  out  of  the  sheath,  they  then  bore  their 
way  through  walls  of  stomach  and  enter  into  a  sort  of  passive  stage,  during  which 
further  development  takes  place.  They  finally  become  distributed  in  the  muscles 
of  the  thorax  and  make  their  way  along  the  fleshy  labium,  to  enter  the  wound  in 
a  person  bitten  by  a  mosquito,  by  way  of  Button's  membrane.  This  development 
takes  about  twenty  days  at  which  time  the  larvae  are  about  }/\§  inch  long  and  have 
an  alimentary  canal.  It  was  formerly  considered  that  the  filarial  worm  of  the 
Philippines  was  a  different  species,  this  from  a  study  of  microfilariae  (F.  phil- 
ippinensis).  Others,  however,  have  considered  the  microfilariae  as  identical  with 
F.  bancrofti.  Recently  Walker  has  published  drawings  and  descriptions  of  four 
adult  filar  iae  in  the  Philippines  which  correspond  to  F.  bancrofti. 

Filaria  perstans. — The  adults  are  found  in  connective  tissue  and  deeper  fat, 
especially  about  the  mesentery  and  abdominal  aorta. 

The  female  is  about  3  inches  (75  mm.)  long;  the  male  is  rarely  found  and  is  less 
than  2  inches  long.  These  worms  are  characterized  by  incurved  tails,  the  extremity 
of  which  has  two  triangular  appendages  giving  a  bifid  appearance.  The  embryos  do 
not  possess  a  sheath  and  have  a  blunt  tail.  The  life  history  is  unknown.  Both 
mosquito  and  tick  have  been  incriminated.  The  embryos  are  always  present  in 


MICROFILARIAE  319 

the  peripheral  circulation — hence  perstans.  There  does  not  seem  to  be  any  symp- 
tomatology. 

It  is  of  historical  interest  that  F.  perstans  was  once  considered  the  cause  of  sleeping 
sickness. 

Onchocerca  volvulus  (Filaria  volvulus).— This  is  a  rather  common  parasite  of 
central  Africa.  The  male  is  about  i%  inches  (35  mm.)  and  the  female  about  5  inches 
long.  The  females  are  so  interlaced  in  the  fibro-cystic  swellings  that  it  is  difficult  to 
determine  their  length.  The  tumors  start  from  the  presence  of  a  worm  in  a  lym- 
phatic. The  tumors  are  easily  enucleated.  Adults  are  striated.  They  are  found  in 
cystic  tumors,  especially  about  the  axilla  and  popliteal  space.  The  cystic  contents 
contain  abundant  sheathless  larvae  about  300/1  long.  It  was  formerly  thought  that 
these  larvae  were  absent  from  the  peripheral  circulation  but  more  recent  investiga- 
tions have  shown  a  sheathless  embryo  in  the  blood  of  patients  with  Onchocerea 
nodules  which  had  the  characteristics  of  those  found  in  the  contents  of  the  nodules. 
Life  history  unknown,  although  it  has  been  suggested  that  a  species  of  Glossina  may 
be  concerned. 

Filaria  demarquayi. — The  habitat  of  this  filarial  worm  is  the  West 
Indies.  The  embryo  has  no  sheath  and  has  a  sharp  tail.  Other  filarial 
species  which  have  been  reported  are  F.  magalhcesi,  F.  ozzardi,  F. 
volvulus,  F.  powellij  and  F.  philippinensis.  A  species  called  F.  gigas 
is  now  considered  to  have  been  only  the  hair  of  the  leg  of  a  fly.  The 
embryos  have  usually  been  given  such  names  as  F.  nocturna,  F.  diurna, 
etc.  Of  course  the  embryos  and  the  parent  should  have  the  same  name. 
It  has  been  proposed  to  designate  these  embryos  the  same  as  the  parent, 
but  with  the  use  of  the  term  Microfilaria  instead  of  Filaria. 

The  points  usually  noted  in  the  description  of  filarial  embryos  are: 

1.  Presence  or  absence  6f  periodicity  of  embryos  in  peripheral 
circulation. 

2.  Presence  or  absence  of  a  sac  sheath  around  the  embryo. 

3.  Accurate  measurements. 

4.  Shape  and  description  of  head  and  tail  ends. 

5.  Character  of  movement. 

6.  Location  of  V  spot  and  break  in  cell  column  in  stained 
specimens. 

KEY  TO  FILARIAL  LARV.E  IN  PERIPHERAL  CIRCULATION 

A .  Sheath  present. 

1.  No  periodicity. 

F.  philippinensis.  Tightly  fitting  sheath;  not  flattened  out  beyond  extremi- 
ties. Tail  is  pointed  and  abruptly  attenuated.  Lashing  progression  move- 
ment. 320  X  6.5;*. 

2.  Periodicity  exhibited. 


320 


THE   ROUND   WORMS 


(a)  Nocturnal  periodicity. 

F.  bancrof  ti  (F.  nocturna) .     Pointed  tail ;  loose  sheath ;  lashing  movement. 
300  X  7.5M-     V  spot  gon  from  head;  break  in  cells  50^  from  head. 

(b)  Diurnal  periodicity. 

F.  loa  (F.  diurna).     Pointed  tail;  loose  sheath;  245  by  7  microns.     V  spot 
60  to  70  microns  from  head,  break  in  cells  40  microns  from  head. 

GENERAL  FILARIAL  KEY 


Adults 

Embryos 

Remarks 

Filaria 
bancrofti. 

Male    40    by    o.i    mm. 
Female  90  by  0.28  mm. 
Smooth  cuticle.     Bulb- 
ous anterior  extremity. 
Occupy    lymphatic 
glands  and  vessels. 

Sheathed,       300       by       7.5 
microns.      Distance     from 
head  to  V.  spot  90  microns; 
to  break  in  cells  50  microns. 
Tail  rather  straight.     Ter- 
minal cells  do  not  fill  up  tail 
end.    Nocturnal  periodicity 
in  peripheral  circulation. 

Transmitted  by  mos- 
quitoes.    Culex  fati- 
gans  and  Stegomyia 
p  s  e  u  d  o  scutellaris. 
Causes  elephantiasis, 
lymph  scrotum,  chy- 
luria,  etc. 

Filaria  loa.      < 

Male    27    by    0.3    mm. 
Female  55  by  0.4  mm. 
Cuticle      tuberculated. 
Anterior  extremity  like 
truncated  cone.     Wan- 
ders   in    subcutaneous 
tissues. 

Sheathed,   240  X  7  microns. 
Distance  from  head  to  V 
spot  65   microns;  to  break 
in  cells  40  microns.     Cork- 
screw  tail    which   is   com- 
pletely filled  up  with  ter- 
minal cells.           Diurnal 
periodicity     in    peripheral 
circulation. 

Transmitted  by 
species  of  a  bit- 
ing fly  —  Chrysops. 
Causes  calabar  swell- 
ings. Worms  often 
visit  ocular  region. 

Filaria 
perstans. 

Male  40    by  0.07    mm. 
Female  75  by  o.i  mm. 
Cuticle     smooth.     Tip 
of  tail  shows   two   tri- 
angular     processes. 
Found    about    root    of 
mesentery. 

Without  sheaths,  200  by  5 
microns.      Posterior      two- 
thirds  tapers  to  blunt  end- 
ing.    Distance   from   head 
to  V  spot  49    microns;  to 
break  in  cells  34   microns. 
Persists  in  circulation  both 
day  and  night. 

Transmitting  agent 
not  surely  known. 
Mosquitoes  and  ticks 
suggested.  No  patho- 
genicity. 

Filaria 
volvulus. 

Male   30   by   0.14   mm. 
Female    usually    frag- 
mented.    Possibly     75 
by   0.36   mm.     Cuticle 
striated.    Found  coiled 
up  in  cyst-like  tumors 
under  skin. 

Without    sheaths.     250    by 
7.5      microns.     Found     in 
cyst-like  spaces  of  tumors. 
Found    also    in    peripheral 
blood  and  lymph  glands. 

Method  of  transmis- 
s  i  o  n  unknown. 
Causes  small  cystic 
tumors,  under  skin  of 
thorax  especially. 

Filaria 
medinensis. 

Male       from       Leiper's 
monkey    22    mm.     Fe- 
male 80  to  90  cm.  long 
by      1.6      mm.      wide. 
Smooth     white     body. 
Anchoring  hook  at  tail 
end.     Female   lives   in 
subcutaneous  tissue  of 
lower  extremity. 

Without      sheaths.      600  X 
20  microns.     Long  slender 
tail.    Cuticle  striated.    Ex- 
truded from  break  in  skin 
of  patient. 

Embryos     swallowed 
by     Cyclops.     Man 
drinks     water     con- 
taining Cyclops. 

THE   WHIP   WORM 


321 


B.  Absence  of  sheath.     None  of  these  exhibit  a  periodicity,  being  continuously  pre- 
sent. 

1.  Blunt  tail — F.  perstans.     200  X  4.5;*. 

2.  Sharply  pointed  tail: 

(a)  F.  demarquayi.     210  X  SM- 

(b)  F.  ozzardi.     215  X  SM- 

(c)  O.  volvulus.     250  —  3oo/i  X  7-5M- 

NOTE. — A  filarial  embryo,  F.  powelli,  reported  once.     It  has  a  sheath,  nocturnal 
-periodicity,  and  is  about  130  X  SM- 


TRICHOTRACHELID^E 

These  have  a  long  thin  neck  and  a  thicker  terminal  portion.  The 
oesophagus  is  of  the  single  row  of  cells  type.  The  anus  is  terminal; 
there  is  only  one  ovary. 

Trichuris  trichiura  (Trichocephalus  dispar).— This  is  usually  called 
the  whip-worm — the  thickened  body  representing  the  handle  and  the 
narrow  neck  the  lash.  It  is  one  of  the  most  common  parasites  in  both 
temperate  and  tropical  climates. 

The  egg  is  very  characteristic  in  having  an  oval  shape  with  knobs  at  either  extrem- 
ity. It  resembles  a  platter  with  handles.  The  male  is  almost  2  inches  long,  and  has 
the  terminal  portion  curled  up  in  a  spiral.  It  has  a  single  terminal  spicule. 

The  female  is  a  little  longer  than  the  male,  and  has  the  terminal  part  in  the  shape 
of  a  comma  instead  of  being  coiled.  The  neck  only  contains  the  oesophagus  which 
is  contained  in  a  groove  in  large  cells  which  form  a  single  row  like  a  string  of  pearls. 
These  cells  play  a  digestion  role.  The  vulva  opens  at  the  upper  end  of  the  thickened 
terminal  end  which  contains  an  intestine  lying  between  the  ovary  and  uterus.  The 
great  powers  of  resistance  of  the  ova  may  account  for  their  general  distribution; 
they  may  live  for  months  under  conditions  of  freezing  and  so  forth.  There  is  no 
intermediate  host.  The  worm  arrives  at  sexual  maturity  in  about  one  month  after 
ingestion.  The  whip-worm  prefers  the  caecum,  but  also  lives  in  the  lower  end  of  the 
ileum  and  the  appendix. 

The  neck  burrows  into  the  mucosa,  and  much  importance  has  been  attributed  by 
the  French  to  the  possibility  of  this  paving  a  way  for  the  entrance  of  pathogenic 
bacteria.  They  do  not  seem  to  produce  serious  symptoms. 

Trichinella  spiralis  (Trichina  spiralis). — The  cause  of  trichinosis  is 
usually  termed  Trichina  spiralis  in  medical  works. 

The  adults  live  in  the  duodenum  and  jejunum;  the  males  are  about  KG  incn 
(1.5  mm.)  long  with  two  tongue-like  caudal  appendages  and  without  a  spicule. 
These  two  lateral  projections  enable  the  male  to  hold  the  female  in  copulation — 
the  cloaca  being  evaginated  to  act  as  a  penis. 

The  females  are  about  M  inch  (3  to  4  mm.)  long.  The  female  gives  off 
embryos  from  the  vulva  which  is  near  the  mouth  end  (viviparous). 


322 


THE   ROUND   WORMS 


These  parasites  can  be  seen  with  an  ordinary  magnifying  glass.  With  higher 
powers  the  oesophagus  has  the  appearance  of  a  serrated  line  instead  of  an  cesophageal 
bulb.  The  male  is  about  40;*  broad  and  has  a  prominent  testicular  enlargement 
filling  the  posterior  extremity.  The  female  is  about  6opt  broad  and  has  a  rounded 
posterior  extremity  with  a  prominent  slit-like  cloaca.  It  is  in  this  posterior  extrem- 
ity that  the  female  increases  in  size  as  she  becomes  filled  with  eggs.  The  vulva  is  in 
the  anterior  third.  After  fertilization  of  the  females  the  males  die,  and  the  females 
bore  into  the  intestinal  mucosa  and  begin  to  produce  embryos  to  the  number  of 
more  than  1000  each.  These  gain  access  to  the  lymph  channels  and  are  distributed 


2.  Stronyytoides      etercoralia. (parasitic.) 

1.  Trichinella     spiralis.  A-Mhenooenetic    female 

B-Rhabditifirm     embryo      , 


7-  Tern  idena       '£i.     b-femole      c-poftericr 
demlnutuS  nirtmity  of  mate,     (spiiules) 
d*e-  anterior  extremities. 


brumpti 


FIG.  74. — Some  of  the  human  nematodes. 

by  the  blood-stream  to  the  striated  muscles.  Embryos  reaching  other  tissues  fail  to 
develop. 

It  is  about  ten  days  before  they  reach  the  muscle.  In  the  muscle  they  become 
encysted  as  the  oval  lemon-shaped  areas  containing  coiled-up  embryos  that  every- 
one is  familiar  with.  These  oval  areas  are  about  450  X  250/1  and  have  a  chitinous 
capsule. 

The  encysted  trichinae  are  found  chiefly  in  the  muscle  fibers  of  the  tongue  and 
diaphragm  and  may  remain  alive  as  long  as  ten  to  twenty  years;  finally,  however, 
the  cyst  undergoes  calcareous  infiltration  and  the  embryo  dies. 

When  uncoiled  the  embryo  is  about  i  mm.  long  with  the  mouth  at  the  attenu- 
ated end.  Among  cannibals  it  would  be  easy  to  keep  the  cycle  going  by  eating 
improperly  cooked  or  raw  human  meat,  the  parasite  being  thus  transmitted. 


TRICHINOSIS 


323 


As  this  would  not  explain  the  transmission  among  civilized  men,  the 
following  is  the  life  history:  Man  obtains  his  infection  from  eating  raw 
pork,  the  embryos  encysted  in  the  muscle  of  the  hog  being  liberated  in 
the  stomach,  and  the  males  and  females  developing  in  the  intestine  as 
above  described.  The  hog  may  gain  his  infection  by  eating  the  meat 
of  other  hogs  or  rats.  These  rats  eat  scraps  of  pork  at  slaughter  houses 
a»d  become  infected.  Being  cannibals,  rats  when  once  infected,  con- 
tinue to  propagate  the  infection.  In  man,  during  the  first  two  or  three 
days  while  the  adults  are  breeding  in  the  intestine,  we  have  gastroin- 
testinal symptoms. 

It  is  during  this  period  or  at  any  rate  before  the  fifth  day  that  purging  may  be  of 
benefit.  About  ten  to  twenty  days  after  infection  the  embryos  begin  to  wander  and 


FIG.  75.— Trichina  spiralis.     (Ziegler.) 

we  have  the  acute  muscle  pains.  In  the  diagnosis  we  should  try  to  obtain  specimens 
of  the  pork  which  has  caused  the  trouble  in  order  to  examine  for  encysted  trichinae, 
or  to  feed  to  white  rats  or  rabbits,  subsequently  examining  the  diaphragm  of  these 
animals  for  encysted  trichince  or  the  intestine  for  adult  trichinae.  Excision  of  a 
small  piece  of  the  deltoid  of  man  may  confirm  the  diagnosis.  The  best  method  is  to 
take  blood  in  3%  acetic  acid,  centrifuge,  and  examine  for  larvae. 

During  the  diarrhceal  stage  we  may  examine  the  stools  for  adult  worms,  in  particu- 
lar dead  males  or  possibly  actively  motile  embryos— these  latter  are  about  90  X  6/i. 

Always  examine  the  blood  for  eosinophilia. 

It  is  well  to  remember  that  the  parts  of  meat  which  trichinae  prefer  (muscle  of 
diaphragm,  of  neck,  etc.)  are  often  used  in  sausage.  Unfortunately  it  is  almost 
impossible  to  detect  the  embryos  in  sausage  meat. 


324  THE    ROUND    WORMS 

STRONGYLID.E 

In  this  family  the  male  has  a  caudal  bursa,  a  prehensile  sort  of  ex- 
pansion at  the  posterior  end  for  copulatory  purposes. 

The  mouth  is  usually  provided  with  six  papillae  and  at  times  with  a 
chitinous  armature.  Those  without  the  chitinous  armature  are  in- 
cluded in  the  subfamily  Strongylinae  (Strongylus,  Trichostrongylus) 
while  those  having  an  armed  mouth  are  in  the  subfamily  Sclerosto- 
minae  (Ancylostoma,  Necator,  Triodontophorus,  (Esophagostoma,  Phy- 
saloptera). 

Eustrongylus  gigas  (Strongylus  renalis). — This  is  the  largest  round 
worm  infecting  man;  it  is  usually  found  in  the  pelvis  of  the  kidney 
(giant  strongyle). 

• 

Two  or  more  worms  may  so  distend  the  kidney  as  to  convert  it  into  a  mere  shell. 
Pain,  haematuria  and  other  symptoms  of  pyelitis,  together  with  the  finding  of  the 
eggs,  make  the  diagnosis.  There  seem  to  be  seven  authentic  and  eight  doubtful 
cases  of  infection  in  man. 

The  females  are  about  40  inches  (i  m.)  long  and  about  Y$  inch  (8  mm.)  in 
breadth  while  the  male  is  about  10  inches  (25  cm.)  long. 

The  collar-like  copulatory  bursa  of  the  male  distinguishes  it  from  Ascaris  as  does 
also  the  dark  red  color.  The  source  of  infection  is  unknown  but  it  has  been  suggested 
that  the  larval  stage  may  exist  in  fish. 

Many  of  the  reported  cases  were  simply  fibrinous  clots  from  ureters  or  wandering 
round  worms. 

The  very  characteristic  ova,  with  gouged-out  oval  depressions,  may  be  found  in 
the  urine,  and  are  diagnostically  confirmatory. 

Strongylus  apri. — This  nematode  is  common  in  the  lungs  of  hogs,  producing  a 
bronchitis  in  young  animals  but  apparently  harmless  for  adult  ones.  It  has  been 
reported  once  from  the  lungs  of  a  six-year-old  boy.  The  male  is  about  i  inch  (25 
mm.)  long  with  two  long  spicules.  The  female  is  about  2  inches  long  and  has  a 
sharply  hooked  posterior  extremity  with  the  vulva  just  beyond  the  bend.  The 
mouth  has  six  lips.  The  eggs  contain  embryos  when  laid. 

Trichostrongylus  instabilis. — This  is  a  small  strongyle  formerly  known  as  Stron- 
gylus subtilis.  The  male  is  about  %  inch  (4  mm.)  long,  and  the  female  about 
Y±  inch  (6  mm.)  Anteriorly  it  tapers  to  a  pointed  head  end  which  is  only 
about  one-tenth  the  thickness  of  the  posterior  extremity.  The  male  has  a  bursa 
and  two  prominent  equal  spicules.  It  has  been  found  in  the  upper  part  of  the  small 
intestine  of  inhabitants  of  Egypt  and  Japan.  It  does  not  appear  to  produce  symp- 
toms. Ova  like  hookworm  ones  (73  to  90  X  42;*).  Ransom  gives  T.  col  ubr  if  or  mis 
as  the  proper  name.  It  is  a  common  parasite  of  sheep  and  goats  in  the  U.  S.  and 
may  exist  in  man  in  such  regions.  Stiles  has  kept  in  mind  the  possibility  of  such 
infections  in  his  Southern  States  hookworm  work  but  has  failed  to  find  cases  in 
man.  The  eggs  are  not  only  larger  than  hookworm  ones  but  show  later  stages  of 
segmentation. 

Triodontophorus  deminutus. — This  is  a  small  round  worm  with  three  forked  teeth 


HOOKWORMS 


325 


taking  origin  from  the  pharyngeal  lobes.  The  collar-like  mouth  orifice  is  made  up  of 
22  rounded  plates  just  inside  the  round  mouth  opening.  They  are  less  than  ^ 
inch  long  and  have  once  been  found  in  the  intestinal  canal. 

(Esophagostoma  brumpti.— Six  young  females  were  found  in  a  cyst  of  the  colon  in 
an  African  negro.  They  were  about  >£  inch  (8  mm.)  long.  The  anterior  end  pre- 
sents an  ovoid  protuberance  with  a  second  cuticular  inflation  just  below  it.  The 
buccal  capsule  is  very  shallow  and  surrounded  by  about  a  dozen  chitinous  plates. 
The  mouth  has  six  papillae. 

This  species  has  recently  been  reported  by  Thomas  in  a  native  of  Brazil. 

Physaloptera  caucasica.— Mouth  with  two  equal  laterally  placed  lips,  each  hav- 
ing three  papillae  and  three  teeth.  The  male  has  a  lancet-shaped  posterior  extremity 
and  is  about  %  inch  long  (14  mm.  by  0.71  mm.).  Female  is  about  i  inch  long  (27 
mm.)  with  a  rounded  tail  end.  Found  only  once  in  the  alimentary  canal  of  a 
native  in  the  Caucasus.  Leiper  has  recently  reported  a  species  P.  mordens  from 
Uganda,  one  case. 

THE  HOOKWORMS  OP  MAN 

The  hookworm  infections  of  man  come  almost  entirely  from  two 
parasites,  Ancylostoma  duodenale,  the  Old  World  species,  and  Necator 
americanus,  which  is  generally  called  the  New  World  species  from  its 
having  first  been  reported  from  the  U.  S.  by  Stiles.  Hookworms 
belong  to  the  class  Nematoda  and  family  Strongylidae. 

Quite  recently  Lane  has  reported  a  new  species,  A.  ceylanicum,  as  having  been 
obtained  from  three  men  in  Bengal,  after  treatment.  This  species  is  the  one  that 
infects  the  civet  cat  in  Ceylon.  So  far  as  we  know  the  other  human  species  belong 
solely  to  man. 

The  male  hookworms  are  a  little  more  than  J£  inch  (9  mm.) 
long  and  the  females  a  little  more  than  J^  inch  (13  mm.)  in  length. 
The  males  can  readily  be  distinguished  by  their  posterior,  umbrella-like 
expansion  or  copulatory  bursa.  The  tail  of  the  female  is  pointed.  The 
vulva  of  A.  duodenale  is  located  in  lower  half  of  the  ventral  surface; 
that  of  N.  americanus  in  upper  half.  The  large,  oval  mouth  of  the  Old 
World  hookworm  has  four  claw-like  teeth  on  the  ventral  side  of  the  buc- 
cal cavity  and  two  knob-like  teeth  on  the  dorsal  aspect.  It  also  has  a 
pair  of  ventral  lancets  below  the  four  ventral  teeth.  One  cannot  make 
out  a  dorso-median  tooth.  In  N.  americanus  the  buccal  capsule  is 
round,  smaller  and  the  ventral  teeth  are  replaced  by  chitinous  plates. 
Dorsally  there  are  two  similar  but  only  slightly  developed  lips  or  plates. 
A  very  prominent,  conical  dorso-median  tooth  projects  into  the  buccal 
cavity.  Through  it  passes  the  duct  of  the  dorsal  cesophageal  gland. 
There  are  also  four  buccal  lancets.  The  copulatory  bursa  of  the  Necator 


326 


THE   ROUND   WORMS 


:left 


americanus  is  also  different,  being  terminally  bipartite  and  deeply  c 
in  the  division  of  the  dorsal  ray,  rather  than  tripartite  and  shallow,  as 
with  A .  duodenale.  The  anterior  extremity  of  A  ncylostoma  bends  in  the 
same  direction  as  the  general  body  curve  while  that  of  Necator  hooks 
back  in  an  opposite  direction  to  the  body  curve.  In  general,  Ancylos- 
toma  is  larger  and  thicker  than  Necator. 


•    .    .    .    i    .    .    .    .    I 

SCALE: 


FiG.76. — la,  Copulatory  bursa  of  Nectar  americanus,  showing  the  deep  cleft 
dividing  the  branches  of  the  dorsal  ray  and  the  bipartite  tips  of  the  branches;  also 
showing  the  fusion  of  the  spicules  to  terminate  in  a  single  barb.  Scale  ^0  mm- 
ib,  Branches  of  dorsal  ray  magnified.  2a,  The  buccal  capsule  of  N.  americanus. 
2b,  The  same  magnified.  3a.  Copulatory  of  Ancylostoma  duodenale,  showing 
shallow  clefts  between  branches  of  the  dorsal  ray  and  the  tridigitate  terminations. 
Spicules  hair-like.  3b,  The  dorsal  ray  magnified.  4a,  The  buccal  capsule  of  A. 
duodenale,  showing  the  much  larger  mouth  opening  and  the  prominent  hook-like 
ventral  teeth.  4b,  the  same  magnified.  $a,  Egg  of  N.  americanus.  sb,  Egg  of 
A .  duodenale.  6a,  Rhabditif orm  larva  of  Strongyloides  as  seen  in  fresh  faeces.  6b, 
Rhabditiform  larva  of  hookworm  in  faeces  eight  to  twelve  hours  after  passage  of 
stool. 

The  name  hookworm  was  given  to  these  nematodes  from  the  hook-like  processes 
of  the  ribs  of  the  rays  of  the  copulatory  bursa.  Dubini  called  the  Old  World  para- 
site Agchylostoma,  properly  Ancylostoma,  on  account  of  the  four  formidable  hook-  or 
claw-like  ventral  teeth  of  the  buccal  capsule.  (<ryx«^0<r,  hook,  and  oro/xa,  mouth.) 

A.  ceylanicum  is  somewhat  smaller  than  A.  duodenale  and  in  the  copulatory  bursa 
of  the  male  we  have  a  deeper  cleft  in  the  dorsal  ray  and  two  rather  long  tips  to  each 


ANCYLOSTOMA  AND  STRONG YLO1DES  327 

branch  instead  of  the  shallow  cleft  and  three  stumpy  processes  of  the  two  branches  as 
in  A.  duodenale. 

Goeze  found  a  hookworm  in  a  badger  in  1782.  He  named  the  parasite  Ascaris 
criniformis.  Froelich,  in  1789,  found  hookworms  in  the  fox  and  called  them  hook- 
worms from  the  hook-like  ribs  of  the  copulatory  bursa.  He  proposed  the  generic 
name  Uncinaria.  Therefore  Uncinaria  belongs  to  the  hookworms  of  the  fox  and 
is  not  valid  for  any  human  species. 

In  1838,  Dubini  found  a  hookworm  as  a  human  parasite.  On  account  of  the 
four  ventral  teeth  projecting  from  the  mouth  he  gave  it  the  name  A  gchylostoma  or 
correctly  Anydostoma. 

Bilharz  and  Griesinger  noted  the  connection  of  the  parasite  with  Egyptian 
chlorosis,  but  it  was  not  until  the  time  of  the  St.  Gothard  tunnel  (1880),  that  the 
importance  of  the  parasite  was  recognized.  Grassi  noted  the  diagnostic  value  of 
the  ova  in  faeces  in  1878.  In  1902,  Stiles  noted  and  described  the  hookworm  found 
in  the  United  States  as  different  and  proposed  the  name  Uncinaria  americana,  later 
changed  to  Necator  americanus.  A.  J.  Smith  had  also  recognized  the  morphological 
differences. 

Hookworms  may  be  found  in  the  small  intestine  (jejunum)  of  man 
in  enormous  numbers.  They  either  produce  their  effects  by  feeding  on 
the  mucosa  or  by  causing  loss  of  blood. 

Life  History. — The  delicate-shelled  eggs  pass  out  in  the  faeces,  and 
in  one  or  two  days  a  rhabditiform  embryo  (200  X  14  microns)  is  pro- 
duced. The  mouth  cavity  of  the  embryo  is  about  as  deep  as  the  diame- 
ter of  the  embryo  at  the  posterior  end  of  the  mouth  cavity;  that  of 
Slrongyloides  is  only  about  one-half  as  deep  as  the  diameter. 

As  a  practical  point,  the  anaerobic  conditions  in  the  intestines  seem  to  prevent 
development  of  the  hookworm  ova  or  at  any  rate  the  absence  of  the  oxygen,  so  nec- 
essary for  the  segmentations  preliminary  to  the  formation  of  the  embryo,  prevents  it. 
Therefore  hookworm  ova  in  freshly  passed  faeces  never  show  other  than  commencing 
segmentation  while  development  of  the  larvae  of  Strongyloides  takes  place  in  the  in- 
testines, so  that  in  freshly  passed  faeces  we  find,  generally,  actively  moving  larvae  or 
at  least  eggs  containing  fully  developed  embryos.  Hookworm  ova  very  rarely  show 
more  than  four  segmentations  or  exceptionally  eight  in  the  freshly  passed  egg. 

In  the  presence  of  oxygen  these  ova  rapidly  develop  into  larvae,  particularly  at  a 
temperature  of  about  27°C.  Beyond  37°C.  and  below  i4°C.  development  does  not 
seem  to  take  place. 

The  rhabditiform  larvae  grow  rapidly  and  by  the  third  day  are  about 
300  microns  long  and  undergo  a  primary  moulting.  By  the  fifth  day 
the  bulb-like  swellings  disappear  and  the  larva  becomes  possessed  of  a 
straight  oesophagus,  thereby  becoming  a  strongyloid  larva.  It  then 
undergoes  a  second  ecdysis  or  moulting,  but  instead  of  casting  off  this 
old  covering,  it  retains  it  as  a  protecting  sheath.  At  this  time  it  ceases 
to  take  food  but  can  move  actively  in  its  sheath  so  that  it  can  crawl  up 


328  THE    ROUND    WORMS 

blades  of  grass  or  vertical  sides  of  mines.  They  can  live  in  this  stage 
for  months,  when  moisture  and  shade  are  present,  but  are  rapidly 
killed  by  drying. 

This  is  the  infecting  stage  in  which  the  larvae  bore  their  way  into  the 
skin,  which  is  the  usual  method  of  infection,  or,  occasionally,  by  enter- 
ing the  mouth  on  vegetables  or  otherwise. 

Looss  thought  that  they  entered  the  skin  by  way  of  the  hair  follicles  but  the  idea 
now  is  that  they  can  bore  into  any  part  of  the  skin.  It  only  requires  a  few  minutes 
for  the  larvae  to  enter  the  skin.  From  the  subcutaneous  tissues  they  effect  an  en- 
trance into  lymphatics  or  veins,  go  to  the  right  heart,  thence  to  lungs.  From  the 
alveolar  capillaries  they  pass  into  the  pulmonary  alveoli,  thence  up  the  bronchi  and 
trachea,  to  pass  out  of  the  larynx  and  then  down  the  oesophagus  to  the  stomach. 
The  larva  loses  its  protecting  sheath  in  the  stomach  and  in  a  few  days  develops  a 
provisional  buccal  capsule. 

By  the  end  of  the  second  week,  after  another  ecdysis,  the  larvae  have  grown  to  be 
about  2  mm.  long  and  130  microns  broad  and  in  about  four  weeks  become  adults,  usu- 
ally in  the  jejunum,  where,  after  fertilization  of  the  females  by  the  males,  the  giving  off 
of  eggs  begins.  The  adults  attach  themselves  to  the  mucosa  of  the  intestine,  feeding 
on  the  deeper  structures  of  the  mucosa,  or  on  the  tissues  of  the  submucosa.  Sambon 
believes  that  the  larvae  can  work  their  way  into  the  jejunum  without  going  there  by 
way  of  the  trachea  and  oesophagus. 

By  providing  an  exit  to  the  trachea,  Fiilleborn  demonstrated  that  in  dogs,  in- 
fected with  the  dog  hookworm,  great  numbers  of  larvae  poured  out  of  the  trachea. 
In  other  dogs  he  stitched  the  oesophagus  to  the  skin  and  noted  larvae  coming  out  of 
these  openings.  In  these  dogs,  with  the  ordinary  channel  obstructed,  infection  did 
occur  with,  however,  only  a  few  worms,  thus  showing  the  truth  of  Sambon's  views 
but  at  the  same  time  demonstrating  the  unimportance  of  such  a  route  of  infection. 

The  mouth  cavity  of  the  embryo  is  about  as  deep  as  the  diameter 
of  the  embryo  at  the  posterior  end  of  the  mouth  cavity;  that  of  Strongy- 
loides  is  only  about  one-half  as  deep  as  the  diameter.  There  is  also  a 
globular  expansion  at  the  bottom  of  the  mouth  cavity  with  hook- 
worm embryos  while  with  Strongyloides  ones  the  passage  to  the  oesoph- 
agus is  funnel-shaped.  Also  the  genital  anlage  of  Strongyloides  is  much 
larger  than  that  of  Ancylostoma. 

A  temperature  of  i°C.  kills  the  eggs  in  twenty-four  to  forty-eight  hours.  After 
moulting  twice,  it  remains  rather  quiescent  but  still  lying  inside  the  discarded  skin. 
It  reaches  this  stage  in  from  four  to  fourteen  days  according  to  the  temperature. 

The  soil  in  the  area  of  the  hookworm-egg-laden  stool  becomes  infested  with  these 
larvae  which  will  even  climb  up  blades  of  grass.  It  is  for  this  reason  that  children 
with  their  bare  feet  are  so  liable  to  infection. 

Laboratory  Diagnosis. — As  a  matter  of  fact  the  diagnosis  is  almost  invariably 
made  by  finding  hookworm  ova  in  the  faeces.  The  eggs  are  oval  and  thin-shelled 
with  a  wide,  clear,  glassy  zone  separating  the  more  or  less  segmented,  granular 


HOOKWORM  OVA  IN  F^CES 


329 


central  portion  from  the  shell.  Formed  stools  are  more  satisfactory  for  examination 
than  the  liquid  ones  resulting  from  a  dose  of  salts.  Put  about  2  drops  of  water  or 
i%  trikresol  solution  in  the  center  of  a  glass  slide  and  emulsify  in  it  as  much  of  the 
faeces  as  is  held  by  the  spatulate  end  of  a  wooden  toothpick.  A  small  piece  of  wood 
or  a  match  stick  will  answer.  These  preparations  can  be  readily  examined  without 
a  cover-glass,  using  a  %-inch  objective,  with  a  i-inch  ocular. 

It  is  usually  stated  that  about  500  worms  must  be  present  for  several  months 
to  produce  symptoms.  Grassi  has  thought  that  the  presence  of  150  eggs  in  o.oi 
gm.  faeces  indicates  the  presence  of  1000  worms,  of  which  25%  would  be  males. 


V)_ 

2 

0 

tt~ 

U-^ 

i- 

0" 

o- 


ORAWN  TO          .C.ALC  x         IOOO 


A.- Median  focus    BrSurf&ce  focus 


ArShowind  f/ 
sides  syif 
metrically 
convex 


Oxyuris 

vermicularis  A.B. 


DrAtypicoJ.  unfertilized 

e,§?    (ftftcr  Looss.lQQ^J 


Strongylus 
subtilis     ' 

(fcf  t«r  Looss, 


-."Without  outer 

t*H  I  •"*  .!"'/(   i*\ Tn^rMOiA  TITIC«      iBf  sJ& 

^^  Embryo 

ry     ,-,  „         „    ^  in  stool 

^^/  An_y          \fl^eri2to48 

A0chylostonicx  r  AgchyibstomS 

Trir*hi  iri  K    ^f^S&T*3^*1'"2^    Jn'froshauxjl        Abolt^«ti9o?S)r 
trtcjP--  Strongyloides 

(Origi 


FIG.  77. — Nematode  ova. 

There  may  be  as  many  as  4,000,000  eggs  in  a  stool.  Bass  has  proposed  the  fol- 
lowing method  for  the  examination  of  faeces  for  ova. 

The  faeces,  which  have  been  made  fluid,  should  be  centrifuged  and  the  supernatant 
fluid  containing  vegetable  debris  poured  off.  The  sediment  contains  hookworm 
eggs.  Then  pour  on  sediment  a  calcium  chloride  solution  of  sp.  gr.  1.050.  Again 
centrifuge  and  decant.  Next  add  calcium  chloride  solution  of  a  sp.  gr.  of  1.250  and 
centrifuge.  This  brings  to  the  surface  the  hookworm  eggs  which  may  be  pipetted 
off.  As  a  rule,  the  finding  of  hookworm  eggs  is  very  easy  without  such  a  technic. 

In  certain  cases,  where  a  microscope  is  not  available,  the  diagnosis  may  be  made 
by  finding  the  worms  in  the  stool  following  a  thymol  treatment. 


330  THE   ROUND    WORMS 

The  presence  of  eosinophilia  is  of  great  assistance  in  diagnosis  but  it  should  be 
remembered  that  not  rarely  severe  cases  of  the  disease  fail  to  show  any  excess  of 
eosinophiles. 

Charcot  Leyden  crystals  are  often  present  in  hookworm  stools. 

It  has  been  claimed  that  where  ordinary  microscopical  examination  for  ova  will 
show  40%  of  infections  and  methods  involving  concentration  55%  that  cultural 
methods  will  show  99%.  A  convenient  method  of  culturing  is  to  make  a  pile  of  filter- 
paper  circles  of  2  inches  diameter  and  about  Y±  inch  high  and  place  in  the  center  of  a 
4-inch  Petri  dish.  Fill  the  dish  with  water  about  to  the  height  of  the  filter-paper 
and  spread  a  thick  layer  of  faeces  on  the  top  of  the  filter-paper  island.  The  larvae 
hatch  out  in  about  six  days  and  swim  out  into  the  clear  surrounding  water.  They 
are  best  found  by  centrifuging  the  fluid  containing  them. 


ASCARID.E 

These  have  three  papillae  or  lips  around  the  oral  cavity,  one  dorsal 
and  two  ventral.  The  male  has  two  equal-length  spicules.  An  in- 
termediary host  is  not  needed  in  the  life  history  of  this  family. 

A 

B 


FIG.  78.— Anterior  extremity  of  Ascaris  lumbricoides;  A,  seen  from  front;  B,  seen 
from  dorsal  surface.     (Tyson  after  Railliet.) 

Ascaris  Lumbricoides. — The  male  round  or  eel  worm  is  from  5  to 
8  inches  (18  cm.)  long  and  the  female  from  7  to  15  inches  (30  cm.)  in 
length.  They  are  grayish  to  reddish  in  color  and  are  from  %  to  Y± 
inch  (5  mm.)  in  diameter. 

It  is  probably  the  most  common  parasite  of  man,  especially  in 
children  and  as  it  does  not  require  an  intermediate  host  infection  takes 
place  through  food  or  drink  or  by  fingers  of  children  who  have  been 
playing  where  soil  pollution  exists. 

The  normal  habitat  is  the  upper  part  of  the  small  intestine,  hence  the  ease  with 
which  they  are  vomited  up.  The  three  papillae-like  lips  with  a  constriction  just 
behind  are  easily  studied  with  a  hand  glass.  The  very  long,  whitish,  convoluted, 
thread-like  tubes  of  the  uterus  lead  to  the  opening  of  the  vulva  anteriorly  and  ven- 
trally.  The  male  has  two  large  lance-like  spicules  which  project  from  a  subterminal 


THE  PIN  WORM  331 

cloaca.  The  posterior  extremity  of  the  male  is  curved  ventrally  and  has  seven 
pairs  of  postanal  papillae. 

The  body  of  the  worm  is  transversely  striated  and  resembles  the  ordinary  earth- 
worm, but  is  more  grayish  than  red.  The  ova  are  very  characteristic  with  a  rough 
mammillated  exterior.  This  at  times  is  shelled  off  and  we  have  a  smooth  egg  which 
may  be  mistaken  for  eggs  of  other  parasites.  The  eggs  leave  the  body  in  the  fseces 
and  after  a  long  time — a  few  weeks  to  several  months,  according  to  temperature — • 
develop  an  embryo  which  remains  in  the  shell  until  swallowed  by  man.  It  is  stated 
that  they  will  remain  alive  for  years.  On  being  swallowed,  the  embryo  leaves  the 
egg  and  we  have  males  and  females  developing  in  the  small  intestine.  In  countries 
where  such  parasites  abound,  as  in  Guam  and  the  Philippines,  the  possibility  of  their 
getting  into  the  peritoneal  cavity  through  operative  measures  on  the  intestine  must 
always  be  thought  of. 

Guiart  considers  it  probable  that  Ascaris  may  suck  blood,  produce  intestinal 
ulcerations  and  bacterial  infections,  and  perforate  intestine.  Their  entrance  into 
bile  ducts  or  into  larynx  (vomited)  must  be  considered. 

At  autopsy  they  may  be  found  perforating  the  appendix  or  even  filling  up  the 
pancreatic  duct. 

Some  think  that  the  symptoms  of  itching  of  nose  and  anus,  vertigo,  or  convulsions 
and  anasrhia  may  be  due  to  a  toxin  secreted  by  the  worm. 

Belascaris  mystax. — This  is  a  very  common  parasite  of  the  dog  and 
cat,  but  is  occasionally  found  in  children.  It  is  much  smaller  than  the 
A.  lumbricoides—  male  is  2  to  3  inches  (5  cm.)  long,  female  4  to  5  inches 
do  cm.)  in  length.  The  parasites  are  characterized  by  the  presence 
of  wing-like  projections  from  the  anterior  end  farrow-like  head).  The 
egg  shells  are  quite  thin. 

Leiper  has  reported  an  infection  with  Toxascaris  limbata  in  an 
Egyptian.  This  is  the  smaller  Ascaris  of  the  dog. 

Other  Ascaridae  reported  from  man  are  A.  texana  and  A.  maritime, 
only  one  case  each. 

Oxyuris  vermicularis. — The  parasite  is  also  known  as  the  pin-worm 
or  seat-worm  and  is  more  frequent  in  children  than  in  adults. 

The  male  is  about  He  inch  (4  mm.)  long  and  the  female  a  little  less  than  ^ 
inch  (12  mm.)  in  length.  The  male  has  an  incurved  tail  with  a  single  spicule  and  the 
female  a  long  tapering  tail.  The  vulva  is  in  the  upper  third. 

These  worms  have  a  clear  slightly  bulbous  Turkish  pipe  mouth-piece-like  projec- 
tion surrounding  the  three-lipped  anterior  extremity.  There  is  a  well-marked  bulb 
oesophagus. 

The  eggs  are  thin-shelled  plano-convex,  and  show  a  coiled-up  embryo.  After 
ingestion  of  eggs,  the  adults  develop  in  the  small  intestine  where  copulation  takes 
place;  the  males  then  die.  The  fertilized  females  go  to  the  caecum  and  colon  where 
they  remain  until  they  reach  maturity.  At  this  time  the  females  wander  to  the  rec- 
tum where  they  either  expel  their  ova  or  themselves  work  their  way  out  of  the  anus. 
This  usually  occurs  at  night,  and  the  scratching  induced  by  the  itching  causes  the 


332 


THE    ROUND    WORMS 


wander 


eggs  to  be  widely  spread  about  the  region  of  the  anus.  The  worms  may  also  wandc 
into  the  vagina,  urethra,  or  under  prepuce.  It  will  be  seen  that  as  a  result  of  the 
scratching,  the  fingers  become  contaminated  with  ova  which  may  be  carried  to  the 
mouth  and  so  cause  a  fresh  infection,  no  intermediate  host  being  required.  The 
examination  of  the  material  under  the  finger  nails  of  children  harboring  this  parasite 
may  show  eggs  under  the  microscope.  A  knowledge  of  the  life  history— the  early 
location  in  the  small  intestine,  and  later  on  in  the  large — shows  that  treatment  should 
be  dual  in  its  direction — enemata  for  the  gravid  female  in  the  rectum  and  santonin 
and  calomel  for  the  young  adults  in  the  small  intestine. 

The  diagnosis  is  preferably  made  by  examining  the  stools  for  the 
white,  thread-like  females  which  are  expelled  after  a  diagnostic  dose  of 
calomel  and  salts,  rather  than  by  searching  for  the  eggs. 

These  females,  which  are  packed  with  embryo  containing  eggs,  may  be  seen 
wriggling  on  the  surface  of  the  freshly  passed  faeces.  In  handling  these  worms 
one  should  be  careful  as  they  are  apt  to  cause  infection  should  the  eggs  get  on  the 
fingers. 

ACANTHOCEPHALI 

These  are  called  thorn-headed  worms  on  account  of  a  proboscis  which  projects 
anteriorly  like  a  little  peg. 

There  are  several  rows  of  hooks  surrounding  this  projection  which  are  directed 
backward  to  enable  the  parasite  to  attach  itself  to  the  intestinal  wall.  The  worm 
absorbs  nourishment  through  the  general  body  wall,  there  being  no  alimentary 
canal  or  mouth.  These  worms  are  common  in  hogs.  The  three-shelled  eggs  are  very 
striking  and  the  intermediate  stage  is  in  June  bugs. 

The  Echinorhynchus  or  Gigantorhynchus  gigas. — This  parasite  is  about  6  inches 
(15  cm.)  long  for  the  male  and  10  to  12  inches  (25  cm.)  for  the  female.  It  has 
transverse  rings  and  resembles  Ascaris  but  is  more  white  in  color.  It  is  said  to  be  not 
uncommon  in  southern  Russia. 

The  Echinorhynchus  or  Gigantorhynchus  moniliformis  might  be  contracted 
by  persons  eating  death-watch  beetles  as  is  sometimes  done  for  the  improvement  of 
the  complexion. 

HIRUDINEI  (LEECHES) 

Hirudo  medicinalis. — This  is  the  leech  used  medicinally  for  the  abstraction  of 
blood.  They  have  a  secretion  which  prevents  coagulation  of  the  blood  so  that  when 
they  are  removed  the  wound  still  continues  to  bleed. 

Limnatis  nilotica. — This  species  has  been  found  in  many  parts  of  northern  Africa 
and,  gaining  access  to  the  stomach  through  drinking  water,  it  wanders  to  the  phar- 
ynx, nares,  and  even  trachea.  Manson  refers  to  a  case  of  obstinate  epistaxis  and 
headache  caused  by  a  leech  in  the  nostril. 

This  leech  is  about  4  inches  long  (8  to  10  cm.)  and  about  %  inch  (i.a  cm.)  broad. 
The  dorsal  surface  is  greenish  brown  with  narrow  orange-brown  borders.  The  young 
leeches  are  only  about  )£  inch  (3  mm.)  long  and  taken  in  with  the  drinking  water 


LEECHES  333 

may  attach  themselves  to  the  surface  of  some  mucous  membrane  and  after  some 
weeks  reach  adult  size. 

Hsemadipsa  ceylonica.— These  are  land  leeches  found  in  India,  Philippines, 
Australia,  and  South  America.  They  are  only  about  i  inch  (25  mm.)  long  and  are 
slender.  They  leave  the  damp  earth  to  climb  shrubs  and  from  there  to  drop  on 
animals  or  man  passing  through  the  forest.  The  bites  are  painless,  but  may  be 
followed  by  ulcers.  They  may  get  into  the  nostrils. 

They  will  even  penetrate  thick  clothing  in  order  to  reach  the  skin. 


CHAPTER  XIX 


Order 


Acarina 


THE  ARACHNOIDS 

CLASSIFICATION  or  THE  ARACHNOIDEA 
Family  Subfamily  Genus 


Trombidiidae 
Gamasidae 

Tyroglyphidae 
Sarcoptidae 
Demodicidae 
Tarsonemidae 


Argasinae 


Species 


Ixodidae 


Ixodinae 


Trombidium 

T.  holosericeum 

Dermanyssus 

D.  gallinae 

Tyroglyphus 

f  T.  farinae 
\  T.  longior 

Sarcoptes 

S.  scabiei 

Demodex 

D.  folliculorum 

Pediculoides 

P.  ventricosus 

(Argas 

!  A.  persicus 
\  A.  miniatus 

Ornithodoros 

O.  savignyi 

Ixodes 

I.  ricinus 

Hyalomma 

H.  aegyptium 

Rhipicephalus 

R.  bursa 

Dermacentor 

f  D.  reticulatus 
\  D.  andersoni 

Margaropus 

M.  annulatus 

Amblyomma 

A.  hebraeum 

Haemaphysalis 

H.  leachi 

Lingua  tula 

L.  rhinaria 

Porocephalus 

P.  constrictus 

Pentastomida 


The  class  Arachnoidea  and  the  class  Insecta  belong  to  the  phylum 
Arthropoda.  This  phylum  contains  a  greater  number  of  species  than 
does  any  other  phylum. 

While  the  lobsters,  crabs  and  water  fleas,  which  belong  to  the  class  Crustacea,  are 
important  zoologically,  they  are  of  very  slight  importance  medically.  Besides  the 
Crustacea  we  have  the  thousand-legged  worms  or  Myriapoda. 

The  different  classes  of  Arthropoda  resemble  the  segmented  worms 
but  have  as  distinction  the  possession  of  jointed  appendages  which 
proceed  from  the  somites  in  pairs.  Some  of  the  pairs  of  limbs  are  for 
locomotion;  at  times,  certain  ones  may  be  specialized  for  food  taking. 

The  somites  or  divisions  of  the  body  have  a  chitinous  exoskeleton. 

334 


MITES  335 

Respiration  takes  place  through  the  medium  of  gills  in  the  Crustacea 
and  by  tracheal  tubes  in  the  Myriapoda,  Arachnoidea,  and  Insecta. 

The  Arachnoidea  have  no  antennae  while  the  Myriapoda  and  Insecta  have  a 
single  pair  of  antennae,  the  former  having  numerous  pairs  of  legs  or  jointed  appen- 
dages while  the  latter  have  only  three  pairs  of  legs.  The  Arthropoda  have  seg- 
mented bodies,  but  they  differ  from  the  worms  in  having  jointed  appendages  for  the 
purpose  of  .taking  in  food  and  moving  from  place  to  place.  They  also  have  an 
exoskeleton  which  is  more  or  less  unyielding  from  the  deposit  of  chitin  in  the  cuticle. 
This  cuticle  is  not  a  true  skin  but  only  a  secretion  of  the  epidermis. 

Within  this  external  skeleton  we  have  a  dorsal  digestive  system  and 
a  ventral  nervous  system. 

THE  ARACHNOIDEA 

The  Arachnoidea  differ  from  the  Insecta  in  having  the  head  and 
thorax  fused  together.  They  also  have  four  pairs  of  ambulatory  appen- 
dages, while  the  insects  only  have  three  pairs.  The  Arachnoidea  never 
have  compound  eyes — these  when  present  being  simple.  Of  the  two 
orders  of  Arachnoidea  of  interest  medically  the  Acarina  is  far  more 
important  than  the  Linguatulida. 


ACARINA 

Of  the  acarines  we  are  chiefly  interested  in  the  mites  and  the  ticks. 
The  acarines  do  not  show  any  separation  of  the  abdomen  from  the 
cephalo-thorax.  A  hexapod  larva  develops  from  the  egg;  this  is  suc- 
ceeded by  an  octopod  nymph  which  differs  from  the  adult  in  not  having 
sexual  organs. 

In  addition  to  the  four  pairs  of  legs  in  the  fully  developed  acarine  there  are  two  other 
paired  appendages,  the  chelicerae,  in  front  of  the  mouth,  and  the  pedipalps  on 
either  side  of  the  mouth. 

Trombidiidae 

These  generally  have  a  soft,  more  or  less  hairy  integument  and  are  often  brightly 
colored.  The  two  eyes  are  often  pedunculated  and  the  chelicerse  are  lancet-shaped 
and  the  palps  project  beyond  the  rostrum  as  claw-like  appendages.  A  tip-like 
appendage  on  the  apical  segment  of  the  palps  is  characteristic.  A  very  common 
and  annoying  member  of  this  family  is  the  hexapod  larva  of  the  Trombidium 
holosericeum.  It  is  usually  designated  Leptus  autumnalis.  Popularly  it  is  termed 
"harvest  mite,"  "red  bug"  or  "jigger."  They  are  found  in  the  fields  in  the 
autumn  and  attack  both  man  and  animals.  The  condition  (itching  and  redness) 


336 


THE    ARACHNOIDS 


produced  is  at  times  called  autumnal  erythema.  There  is  a  Trombidium  in  Mexico 
which  has  a  predilection  for  the  skin  of  the  eyelids,  prepuce,  and  navel.  The  Kedani 
mite,  an  orange-red  larval  mite  about  250  by  125  microns  is  believed  by  the  Japanese 
authorities  to  bring  about  infection  with  Japanese  river  fever  or  Tsutsugamushi, 
as  the  result  of  transmitting  either  a  bacterium  or  protozoon  by  its  bite.  The 
disease  somewhat  resembles  typhus,  although  an  eschar  at  the  site  of  the  bite  and 
lymphatic  involvement  is  present. 

Gamasidae 

Of  the  Gamasidae,  which  generally  have  a  hard  leathery  body  and  styliform 
piercing  chelicerae,  delicate  five  jointed  palps  and  styliform  hypostome,  only  the 
Dermanyssus  gallina  is  of  interest.  This  mite  infests  chicken-houses  and  sucks  the 


FIG.  79. — Arachnoidea  exclusive  of  ticks.  (ia)  Sarcoptes  scabiei,  female;  .(ib)  S.- 
scabiei,  male;  (2)  Demodex  foUiculorum;  (3)  Trombidium  akamushi,  hexapod  larva 
(Kedani  mite);  (4)  Trombidium  holosericeum  larva  (Leptus);  (5)  Dermanyssus 
gallina;  (6)  Tyroglyphus  longior;  (7 a)  Pediculoides  ventricosus,  male;  (76)  P.  ventri- 
cosus,  young  male;  (7^)  P.  ventricosus  impregnated  female;  (8)  Porocephalus  armll- 
latus;  (90)  Linguatula  serrata,  female;  (96)  L.  serrata,  larva. 

blood  of  the  inmates.  They  will  also  attack  man.  Poultrymen  may  be  troubled 
with  a  sort  of  eczema  on  the  backs  of  the  hands  and  forearms,  similar  to  scabies, 
resulting  from  bites  by  these  mites.  They  measure  350  X  650^1.  They  have  no 
eyes. 

Tyroglyphidae 

Mites  of  this  family  live  on  cheese,  flour,  dried  fruits,  etc.  They  are  small, 
without  eyes,  and  have  a  smooth  skin  and  a  cone-like  appearance  of  the  mouth 


THE  ITCH  MITE  337 

parts  which  are  largely  formed  by  the  chelate  cheliceras.  They  are  chiefly  of 
importance  because  of  their  being  occasionally  found  in  urine,  faces,  etc.,  and  being 
striking  objects,  the  question  of  pathogenicity  arises.  The  T.  longior  has  been 
associated  with  intestinal  trouble  (probably  a  coincidence,  patient  having  eaten 
cheese  containing  these  mites). 

Glyciphagi  are  found  in  sugar  and  are  the  cause  of  what  is  known  as  "grocers' 
itch."  Rhizoglyphus  parasiticus  is  reported  to  be  the  cause  of  an  itch-like  affection 
of  the  feet  of  coolies  on  tea  plantations.  To  distinguish:  the  dorsum  of  Glyciphagus 
is  hairy  or  plumose;  Tyroglyphus  has  both  claws  and  suckers  on  tarsi,  while  Rhizo- 
glyphtis  has  only  claws. 

Sarcoptidae 

These  are  small  eyeless  mites  with  a  transversely  striated  cuticle.  They  live 
on  the  epidermis  of  man  and  various  animals.  The  rostrum  is  chiefly  made  up  of 
chelate  chelicerae  with  quite  short  three  jointed,  rather  adherent  palpi.  It  is  the 
female  that  makes  the  tunnels  in  the  skin  between  the  fingers,  on  penis,  flexor  surface 
of  forearm,  etc.  The  male  dies  off  after  copulation.  The  female  passes  through  four 
stages:  i.  larva;  2.  nymph;  resembles  adult,  but  has  no  sexual  organs;  3.  the  pubes- 
cent female;  4.  the  egg-bearing  female.  A  pair  of  itch  mites  may  produce  1,500,000 
descendants  in  three  months.  Transference  of  eggs,  larvae  or  pubescent  females 
does  not  seem  to  transmit  scabies.  It  is  the  egg-laden  female  only.  The  human 
itch  mite,  Sar copies  scabiei,  is  an  oval  mite,  the  male  is  250  X  150/^5  the  female  is 
about  400  X  SOOJL*.  Besides  the  difference  in  size,  the  male  may  be  distinguished 
from  the  female  by  the  fact  that  the  third  and  fourth  pairs  of  legs  in  the  female  have 
bristles,  but  in  the  male,  the  fourth  pair  has  suckers.  The  tunnels  made  by  the 
female  have  the  egg-bearing  female  at  the  blind  end;  scattered  all  along  are  fasces, 
eggs,  larvae;  the  eggs  being  next  the  mother  and  the  more  mature  young  at  the  en- 
trance to  the  gallery.  A  diagnosis  can  be  made  from  the  finding  of  either  eggs  or 
larvae.  The  eggs  are  140;*  long  and  hatch  out  in  four  to  five  days.  A  female  becomes 
mature  in  about  two  weeks. 

In  treating  itch  with  sulphur  preparations  the  adult  females  and  immature  itch 
mites  are  killed;  the  eggs,  however,  are  not  affected.  Hence  a  second  treatment 
about  ten  days  after  the  first  is  necessary  to  kill  the  young  mites,  which  have  devel- 
oped subsequent  to  the  first  treatment.  Different  animals  have  different  species  of 
itch  mites. 

Demodicid®  (Hair  Follicle  Mites) 

Demodex  folliculorum. — This  is  a  vermiform  acarine  about  400 JU  long;  the  eggs 
are  about  75/z  long;  they  chiefly  live  in  the  sebaceous  glands  of  nose  and  forehead. 

Tarsonemidae 

This  acarine  family  shows  a  complete  sexual  dimorphism.  The  Pediculoides 
ventricosus  is  oval  and  about  125  X  75M  for  the  male  which  has  claws  at  the  extremi- 
ties of  the  anterior  and  posterior  pairs  of  legs;  the  two  other  pairs  have  booklets  and 
a  sucking  disc.  The  female  is  about  twice  as  long  but  of  the  same  breadth  as  the 
male,  and  has  claws  only  on  the  anterior  legs. 


338  THE   ARACHNOIDS 

The  chelicerae  are  needle  like  with  inconspicuous  palps  and  the  front  and  rear 
pairs  of  legs  are  widely  separated.  The  gravid  female  is  like  a  ball  and  is  about 
looofA  in  diameter. 

They  live  on  wheat  and  may  be  found  in  wheat  straw,  which,  if  handled,  may  be 
followed  by  a  severe  skin  eruption  with  an  irregular  fever. 

Ixodidae 

This  family  of  the  Arachnoidea  is  one  of  great  medical  interest  and 
of  growing  importance.  It  has  recently  been  proposed  to  raise  the 
ticks  to  a  superfamily,  Ixodoidea  and  to  divide  it  into  the  families 
Argasidae  and  Ixodidae. 

While  only  proven  the  intermediary  hosts  in  the  case  of  the  organism  of  African 
tick  fever  and  the  recently  discovered  cause  of  spotted  fever  of  the  Rocky  Moun- 
tains, there  is  considerable  speculation  as  to  the  possibility  of  blackwater  fever  being 
due  to  a  Babesia  (Piro plasma}.  Piroplasmata  of  animals  seem  to  be  invariably 
transmitted  by  ticks. 

Very  important  diseases  due  to  these  small  pear-shaped  organisms  within  red 
cells  are  known  for  various  animals,  the  best  known  being  that  of  cattle  in  Texas 
and  known  as  Texas  fever.  Other  piroplasmata  diseases  are  Rhodesian  fever 
(cattle),  heart  water  (sheep),  and  malignant  jaundice  of  dogs.  In  these  diseases 
there  are  pathological  features  which  resemble  blackwater  fever  of  man. 

It  is  of  interest  to  note  that  it  was  with  the  transmission  of  Texas 
fever  through  an  intermediate  host  (the  tick)  that  Smith  and  Kilborne 
(1889-1893)  established  the  zoological  principle  of  transmission  of 
disease  through  arthropod  intermediary  hosts.  This  led  up  to  the 
work  on  malaria,  yellow  fever,  etc. 

Ticks  differ  from  insects  in  having  four  pairs  of  legs,  only  two  pairs  of  mouth 
parts,  and  no  antennae.  They  differ  from  other  acarines  in  having  a  median  probe- 
shaped  puncturing  organ,  the  hypostome,  which  is  beset  with  numerous  teeth 
projecting  backward,  and  in  possessing  stigmal  plates.  The  head,  or  capitulum, 
or  rostrum,  is  the  part  which  projects  anteriorly  from  the  body.  This  carries  the 
piercing  parts  which  are  the  single  hypostome  or  dart  and  a  pair  of  piercing  chitinous 
structures,  the  chelicerae  which  lie  above  the  hypostome.  As  a  sheath  for  these 
delicate  biting  parts  we  have  a  segmented  pair  of  palpi  or  pedipalps.  The  mouth 
is  a  slit  between  the  chelicerae  and  hypostome. 

Two  depressed  pitted  areas  on  the  dorsal  surface  of  the  capitulum  in  the  adult 
female  are  known  as  porose  areas.  Very  important  structures  are  the  stigmal 
plates.  These  are  striking  mosaic-like  areas  which  are  located  just  posterior  to 
each  hind  leg  in  the  Ixodinae  and  between  the  third  and  fourth  legs  in  the  Argasinae. 
As  the  greatest  confusion  exists  as  to  the  classification  of  ticks,  Dr.  Charles  W. 
Stiles  has  now  in  hand  a  system  of  classifying  ticks  according  to  the  appearance 
of  these  plates  as  seen  under  the  high  power  of  a  microscope.  There  is  great  varia- 


TICKS  339 

tion  in  the  outline  and  general  picture  of  these  stigmal  plates  in  the  different  species. 
The  stigmal  orifice,  the  opening  of  the  tracheal  system,  is  in  the  center.  The 
Ixodinae  have  a  scutum-  or  shield-like  chitinous  structure  on  the  dorsal  surface.  It 
covers  almost  the  entire  back  of  the  tick  in  the  male  and  only  a  small  portion  ante- 
riorly in  the  female.  The  genital  opening  is  toward  the  anterior  part  of  the  ventral 
surface.  The  anus,  with  anterior  or  posterior  anal  grooves,  is  near  the  posterior 
third  of  the  venter.  The  legs  have  six  segments,  the  coxa  being  flattened  out  on  the 
surface  of  the  body  and  the  terminal  tarsus.ending  with  a  pair  of  hooks  and  at  times 
with  a  pulvillus.  The  nymph  has  stigmal  plates  but  has  no  genital  opening  while 
the  larva  has  neither  genital  apertures  nor  stigmal  orifice. 

Life  History  of  Ticks. — This  varies  greatly  according  to  the  sub- 
family, genus,  and  species.  The  female  Ornithodoros  samgnyi  lays 
about  140  eggs.  The  larva  does  not  leave  the  egg,  but  moults  inside, 
and  finally  emerges  as  an  eight-legged  nymph.  It  lives  in  the  dust  in 
the  cracks  of  the  native  huts  and  comes  out  at  night  to  feed  on  the  sleep- 
ing natives.  As  the  possibilities  for  destruction  are  not  so  great  as  with 
many  Ixodinae  the  necessity  for  thousands  of  eggs  is  not  imperative  for 
the  continuation  of  the  species  as  with  the  Ixodinae.  With  some  of 
the  Ixodinae  the  females  lay  from  5000  to  20,000  eggs  during  several 
days  or  weeks  and  then  die.  The  eggs  are  preferably  deposited  near 
grass.  The  egg  stage  lasts  from  two  to  six  months,  when  the  six-legged 
larva  ("seed  tick")  emerges.  It  crawls  up  a  blade  of  grass  and  gets  on  a 
passing  animal.  After  feeding,  or  at  times  without  taking  nourishment, 
the  larva  drops  to  the  ground,  and  changes  to  the  pupal  stage  which  has 
four  pairs  of  legs.  The  pupa  crawls  up  a  blade  of  grass  and  gets  on  a 
passing  animal  (the  second  host).  Feeding,  it  falls  to  the  ground  where 
it  remains  eight  to  ten  weeks.  It  moults  and  develops  into  an  adult 
tick.  These  males  and  females  gain  access  to  a  third  animal  host— 
the  males  fecundate  the  females,  after  which  the  female  gorges  herself 
with  blood;  afterward  dropping  off  the  animal  and  laying  eggs.  With 
some  ticks  fewer  hosts  suffice. 

Cleland  has  noted  reports  of  serious  symptoms,  chiefly  cardiac  and  visual,  from 
the  bite  of  ticks  in  Australia  (Ixodes  holocydus).  This  is  exceptional,  however,  as 
the  symptoms  following  the  bites  of  such  ticks  are  only  those  of  skin  irritation. 

Classification  of  Ixodidae 

Subfamily  Argasinse. — Head  concealed  by  body  when  viewed 
dorsally.  No  scutum.  Stigmal  plates  between  third  and  fourth  legs. 
Adults  have  no  suckers  (pulvillus)  beneath  claws.  Slight  sexual 
dimorphism.  Anus  near  middle  of  venter.  Skin  rough. 


340 


THE   ARACHNOIDS 


Genus  Argas. — Body  narrow  in  front.  Margins  thin  and  acute.  No  eyes. 
The  A.  persicus  (Miana  bug)  of  Persia  has  been  supposed  to  be  concerned  in  the 
transmission  of  a  serious  disease.  Rostrum  some  distance  from  anterior  margin. 
It  is  also  called  the  fowl  tick  and  transmits  fowl  spirillosis. 

Genus  Ornithodoros. — Margins  of  body  rounded.  Skin  has  many  irregular 
tubercles.  Rostrum  even  with  anterior  margin  so  that  ends  of  palpi  slightly  project. 
It  is  the  intermediate  host  of  Spirochata  duttoni.  (South  African  tick  fever.) 

0.  moubata  is  very  common  in  Africa  living  in  cracks  in  mud  floors  and  bites 
severely  the  sleeping  natives.  The  larva  makes  its  first  moult  inside  the  egg  so 
that  it  shows  four  pairs  of  legs  when  it  emerges.  Christy  thinks  it  may  transmit 
Filaria  perstans. 

Tick  Fever. — With  tick  fever  the  epidemiology  rests  upon  the 
life  history  of  the  tick  0.  moubata.  This  tick  infests  the  rest  houses 


FIG.  80. — Ornithodoros   moubata.     (Murray  from   Doflein.) 

along  the  route  of  travel,  hiding  in  the  crevices  of  floors  and  walls  during 
the  day  and  coming  out  at  night  to  bite  the  sleeping  inmates.  The 
feeding  occupies  a  long  time,  more  than  an  hour.  Both  sexes  bite  man. 
The  female  lays  about  100  eggs,  from  which  a  nymph  emerges  in  about 
twenty  days.  The  larval  stage  takes  place  in  the  egg.  Shortly  after 
emerging  the  nymphs  suck  blood.  An  important  fact  is  that  the  female 
transmits  the  spirochaete  to  its  ova,  so  that  the  nymphs  may  transmit 
the  disease. 

Natives  seem  to  sutler  severely  from  tick  fever  in  childhood  but  in  adult  life 
possess  a  sufficient  degree  of  immunity  so  that  the  disease  shows  itself  in  a  very 
mild  form  in  those  harboring  spiroch<etes.  Ticks  can  be  infected  by  these  carriers. 
In  some  of  the  rest  houses  50%  of  the  ticks  may  be  infected.  While  the  tick  does  not 


TICK   FEVER 


341 


00 


342  THE   ARACHNOIDS 

tend  to  leave  its  habitation  it  may  be  transported  in  the  bundles  of  native  porters. 
The  transmitting  agent  of  the  north  African  relapsing  fever  and  probably  of  the 
Indian  type  is  the  louse.  The  body  louse  deposits  about  75  eggs  in  the  clothes  of  the 
host,  which  hatch  out  in  about  four  days  and  become  adults  in  about  two  weeks.  The 
head  louse  deposits  its  eggs  or  nits  on  the  hair  of  the  host's  head.  (See  Pediculus.) 
O.  savignyi  has  two  pairs  of  eyes  near  base  of  mouth  parts. 

Subfamily  Ixodinae. — Mouth  parts  project  in  front*  of  body  when 
viewed  dorsally.  Scutum  present.  Stigmal  plates  posterior  to  fourth 
pair  of  legs.  Adults  have  suckers  beneath  claws.  Skin  finely  striated. 

Anus  behind  middle  of  venter. 

Sexual  dimorphism  marked.  Male  has  well- developed  scutum; 
female  has  porose  areas. 

Section  Ixodae. — Transverse  recurved  preanal  groove  in  female.  Male  has  ventral 
surface  covered  with  chitinous  plates.  No  eyes.  Genus  Ixodes. 

Ixodes  has  long  rostrum  with  slender  palpi — palpi  narrow  at  base,  leaving  gap 
between  them  and  hypostome. 

Section  Rhipicephalus. — No  preanal,  but  postanal  groove  in  female.  Ventral 
surface  of  male  without  adanal  plates  in  Dermacentor,  Hcemaphysalis^  Aponomma 
and  Amblyomma,  but  with  one  or  two  pairs  in  Hyalomma,  Rhipicephalus  and 
Margaropus. 

In  the  genera  Hyalomma,  Aponomma  and  Amblyomma  the  palpi  are  long  and 
slender  and  of  about  uniform  width  of  segments. 

In  Hyalomma  the  segments  of  palpi  are  of  about  equal  length.  In  Aponomma 
and  Amblyomma  the  second  palpal  segment  is  much  longer  than  the  others.  Ambly- 
omma differs  from  Aponomma  in  being  very  ornate  and  in  having  eyes. 

In  the  genera  Hcemaphysalis,  Dermacentor,  Rhipicephalus,  and  Margaropus  the 
palpi  are  short. 

Hcemaphysalis  has  very  broad  rostrum,  triangular  palpi,  and  no  eyes.  Derma- 
centor has  a  square  rostrum  with  short  thick  palpi,  the  second  and  third  joints  being 
as  broad  as  long.  Dermacentor  andersoni  transmits  spotted  fever  of  the  Rocky 
Mountains — not  D.  reticulatus.  The  most  important  characteristic  of  the  genus 
Dermacentor  is  the  large  size  of  the  coxae  of  the  fourth  pair  of  legs. 

Rhipicephalus  has  palpi  without  transverse  ridges  and  comma-shaped  stigmal 
plates.  The  stigmal  plates  of  Margaropus  are  nearly  circular  and  the  palpi  have 
acute  transverse  ridges  externally.  Margaropus  annulatus  transmits  Texas  fever 
of  cattle.  This  tick  is  also  called  'Boophilus  bovis  or  B.  annulatus.  Some  authors 
term  it  Rhipicephalus  annulatus.  Larvae  developing  from  eggs  of  female  ticks 
which  have  fed  on  cattle  infected  with  Texas  fever  transmit  the  disease  which  is  due 
to  a  protozoon  Babesia  bigemina. 

PENSTASTOMIDA  (TONGUE  WORMS) 

These  are  vermiform  acarines  more  or  less  distinctly  annulated.  They  have 
retractile  hooks  at  either  side  of  the  elliptical  mouth. 


TONGUE  WORMS  343 

If  the  hooks  are  to  be  considered  not  as  degenerated  legs  but  antennse  and  palpi, 
then  there  is  no  vestige  of  legs  in  the  adult.  The  sexes  are  separate. 

Linguatula  rhinaria. — This  has  been  observed  in  man  both  in  larval  and  adult 
stages. 

The  male  is  white  and  about  Y±  inch  long  while  the  female  is  about  4  inches 
long,  tadpole  shape,  yellowish  in  color,  and  has  about  90  segments,  lives  in  the 
nasal  cavity  and  frontal  sinus  of  dogs,  rarely  in  horses  and  sheep,  and  very  rarely 
in  man. 

The  female  lays  embryo-containing  eggs  which,  gaining  freedom  through  the 
nasal  mucus,  are  swallowed  by  various  animals.  A  larva  develops  which  bores  its 
way  through  the  gut  and  encysts  in  the  liver  or  mesenteric  glands.  After  several 
moultings,  they  work  their  way  again  to  the  intestines  and  so  get  out  of  the  body 
of  their  host;  or  they  may  wander  to  lungs  and  trachea  and  either  escape  or  take  up 
their  position  in  the  nostrils  to  become  adults  and  produce  eggs.  Consequently, 
one  animal  may  act  as  intermediate  and  definitive  host  or  these  cycles  may  take 
place  in  distinct  animal  hosts. 

The  larval  form  (^  inch)  is  far  more  common  in  man  than  the  adult.  Symptoms 
are  referred  to  liver  in  both  larval  and  adult  stage,  and  epistaxis  and  nasal  symptoms 
for  adult  stage  only. 

Porocephalus  constrictus. — The  adult  form  P.  moniliformis  lives  in  the  lungs  of 
snakes  and  the  eggs  are  probably  ingested  by  drinking  water.  These  eggs  develop 
into  a  curled-up,  ringed  larva,  about  ^  inch  long  with  23  rings,  which  is 
encysted  especially  in  the  liver  or  lungs.  These  escape  and  are  swallowed  by  the 
snakes,  their  definitive  hosts. 

While  in  the  liver  or  lungs  of  man  the  patient  may  have  signs  of  bronchitis,  hep- 
atitis or  peritonitis.  Cases  usually  only  discovered  at  postmortem.  Parasites, 
however,  might  possibly  be  found  in  sputum  or  faeces. 


CHAPTER  XX 
THE  INSECTS 

CLASSIFICATION  OF  THE  CLASS  INSECTA 


Order               Family 

Subfamily              Genus 

Species 

Siphunculata 

Pediculidae 

1  Pediculus 

P.  capitis 
P.  vestimenti 

[  Phthirius 

P.  pubis 

Rhynchota 

Acanthiidae 

Acanthia 

A.  lectularia 

(Hemiptera) 

Reduviidae 

Conorhinus        < 

C.  megistus 
C.  sanguisuga 

Pulex 

P.  irritans 

Xenopsylla 

X.  cheopis 

Pulicinae     - 

Ceratophyllus 

C.  fascia  tus 

Siphonaptera  • 

Pulicidse 

Ctenocephalus 

C.  serraticeps 

Ctenopsylla 

C.  musculi 

Sarcopsyl-      Sarcopsylla 

S.  penetrans 

linae 

Simulidae 

Shnulium 

S.  reptans 

(buffalo  gnats) 

Psychodidae 

Phlebotomus 

P.  papatasii 

(moth  midges' 

Chironomidae 

Ceratopogon 

C.  pulicaris 

(midges) 
Culicidse 

'Culicin*     /Stegomyia 
\  Culex 

S.  calopus 
C.  fatigans 

Anophe-         Anopheles 

A.  maculipennis 

linae 

Tabanus 

T.  bovinus 

Tabanidae 

Haematopota 

H.  pluvialis 

(horseflies) 

Pangonia 

P.  beckeri 

Chrysops 

C.  dispar 

Diptera 

Glossina             < 

G.  palpalis 

1 

G.  morsitans 

Stomoxys 

S.  calcitrans 

Muscidas 

Musca 

M.  domestica 

Auchmeromyia 

A.  luteola 

Calliphora 

C.  vomitoria 

Lucilia 

L.  caesar 

Chrysomia 

C.  macellaria 

(screw-worm) 

Sarcophagidae 

(Sarcophaga 
Ochromyia 

S.  carnaria 
O.  anthropo- 

haga 

(Estridae 

{Dermatobia 
Hypoderma 

D.  cyaniventris 
H.  diana 

344 


LICE  345 

INSECTA 

The  class  Insecta  has  one  pair  of  antennae,  three  pairs  of  mouth  parts 
(the  fused  labium  being  considered  as  one  pair),  and  three  pairs  of  legs. 
They  have  three  divisions  of  the  body — head,  thorax,  and  abdomen. 

The  head  carries  the  antennae  and  mouth  parts;  the  thorax,  which  is  divided 
into  the  pro  meso  and  meta  thorax,  carries  upon  the  ventral  surface  of  each  thoracic 
segment  a  pair  of  legs  and  on  the  dorsal  surfaces  of  the  two  posterior  segments  a 
pair  of  wings.  The  abdomen  does  not  support  appendages.  The  air  is  supplied 
by  means  of  tracheae — branching  breathing  tubes  which  have  external  openings  or 
stigmata.  The  tracheae  are  stiffened  by  spiral  chitinous  bands.  The  Malpighian 
tubules  are  excretory  organs  of  the  alimentary  system  and  excrete  nitrogenous 
waste  material.  Insects  have  two  pairs  of  wings,  the  second  pair  of  which  is  fre- 
quently rudimentary  and  shows  simply  as  knob-like  projections.  These  are  termed 
halteres  or  balancers.  In  some  insects  both  pairs  of  wings  are  rudimentary,  as  in 
Siphonaptera. 

Where  insects  show  metamorphosis  we  have  voracious  worm-like  larvae  coming 
out  of  eggs;  these  larvse  are  succeeded  by  a  quiescent  no nf ceding  encased  pupa 
which  finally  develops  into  an  imago  or  fully  developed  insect.  An  insect  which  does 
not  present  this  developmental  cycle  shows  incomplete  metamorphosis.  Of  the 
class  Insecta  only  the  Siphunculata,  Rhynchota,  Siphonaptera,  and  Diptera  are  of 
special  importance. 

SIPHUNCULATA  (ANOPLURA) 

These  are  small  dorso-ventrally  flattened  wingless  insects  not  showing 
metamorphosis. 

The  Pediculidae 

In  this  family  there  are  no  wings  and  there  is  no  metamorphosis. 
The  acorn-shaped  eggs  (nits)  are  deposited  on  hairs  of  the  host. 

Pediculus  capitis. — The  female  is  about  %  inch  (3  mm.)  long;  the  male 
smaller.  They  vary  in  color  according  to  the  color  of  the  hair  of  the  host.  The  eggs 
are  deposited  on  the  hairs  of  the  head  in  number  of  60  which  hatch  out  in  about  six 
days.  The  thorax  is  as  broad  as  the  abdomen.  The  male  louse  is  rounded  off 
posteriorly  and  shows  a  dorsal  aperture  for  a  pointed  penis,  while  the  female  is 
recognized  by  a  deep  notch  at  the  apex  of  the  last  abdominal  segment.  There  seems 
to  be  a  marked  preference  exhibited  by  lice  for  their  own  peculiar  racial  host.  It  has 
been  suggested  that  this  might  account  for  certain  peculiarities  in  infection  where 
different  races  were  living  together  and  under  similar  conditions  as  to  food  and 
environment,  and  yet  only  one  race  contracts  the  disease  (beriberi).  The  head  louse 
has  been  found  to  harbor  leprosy  bacilli  when  living  on  a  leper. 

Pediculus  vestimenti. — This  louse  lives  about  the  neck  and  trunk  and  deposits 
its  eggs  in  the  clothing.  They  number  about  75  and  hatch  out  in  three  or  four  days 
and  become  mature  in  about  two  weeks.  Unlike  the  fleas  there  is  no  grub  stage. 


346 


THE   INSECTS 


It  is  almost  twice  the  size  of  the  P.  capitis  and  the  abdominal  seg- 
ment is  broader  than  the  thorax.  The  abdomen  is  less  markedly 
festooned  than  that  of  P.  capitis;  is  less  hairy  and  contains  8  segments 
as  against  7  for  P.  capitis. 

It  has  recently  been  shown  to  transmit  typhus  fever  and  more  recently  Nicolle 
has  demonstrated  it  as  a  carrier  of  relapsing  fever,  the  spirochaetes  being  introduced 
by  the  material  from  the  crushed  louse  being  rubbed  into  the  wound  by  the  scratch- 
ing of  the  victim  (just  as  with  the  flea  in  plague)  and  not  by  the  bite  itself.  The 
dog  louse  as  well  as  the  dog  flea  serves  as  an  intermediary  host  for  Dipylidium. 


FIG.  82. — Siphunculata  and  Rhynchota.  i.  Pediculus  capitis.  2,  Pediculus 
vestimenti.  2 a.  Protruded  rostrum  of  Pediculus.  3.  Phthirius  pubis.  4.  Acanthia 
lectularia.  5.  A.  rotundata.  6.  Conorhinus  megistus. 

Phthirius  pubis. — This  louse  is  popularly  known  as  the  crab  louse.  The  female  is 
little  more  than  %5  inch  in  length,  and  the  male  a  trifle  less.  They  are  almost 
square.  The  second  and  third  pair  of  legs  are  supplied  with  formidable  hooks. 
They  have  a  preference  for  the  white  race  and  live  about  the  pubic  region.  The 
female  lays  about  a  dozen  eggs,  which  hatch  out  in  about  a  week. 


RHYNCHOTA 

The  Rhynchota  are  insects  possessing  a  sucking  beak  in  which  the 
lower  lip  forms  a  long  thin  tube  or  rostrum  which  can  be  bent  under  the 


BEDBUGS 


347 


head  or  thorax.     Inside  this  tube  are  biting  parts— mandibles  and  max- 
illae.    The  metamorphosis  in  this  order  is  not  marked. 

They  have  no  palpi.  The  lower  lip  or  labium  or  beak  has  its 
edges  curved  to  form  the  tube  and  it  is  only  covered  by  the  labrum  at 
its  base.  With  the  Diptera  the  labrum  goes  into  the  formation  of  the 
sucking  tube.  The  mandibles  and  maxillae  are  bristle-like  structures 
serrated  at  the  tip.  The  mandibles  are  grooved  internally  and  form 
when  apposed  a  tube  for  blood. 


The  Acanthiidae 

These  have  a  flattened  body,  a  three-jointed  rostrum,  and  four- 
jointed  antennae.     Their  wings  are  atrophied. 


FIG.  83. — Fleas,  bedbugs  and  ticks.  A,  Lcemopsylla  cheopis;  B,  P.  irritans; 
C,  Ctenopsylla  musculi;  D,  bedbug;  E,  cross  section  of  rostrum  of  Ornithodorus; 
F.  longitudinal  section  of  Ornithodorus. 

Acanthia  lectularia  (Cimex  lectularius). — This  is  the  cosmopolitan  bedbug. 
It  measures  about  K  by  M  inch  (5  by  3  mm.).  It  is  of  a  brownish-red  color. 
The  most  conspicuous  feature  of  the  bedbug  is  the  long  proboscis  continuous  with 
the  dorsal  integument  of  the  head  and  tucked  under  the  ventral  surface.  There  are 
two  prominent  eyes  and  two  four-jointed  antennae.  There  are  eight  abdominal 
segments.  The  bedbug  lives  in  cracks  and  crevices,  especially  about  beds.  It  is 
said  they  can  migrate  from  house  to  house.  At  any  rate,  they  are  frequently  trans- 


348  THE   INSECTS 


The 


ferred  with  wash  clothes.  They  have  a  penetrating  odor  when  crushed.  The 
female  deposits  about  50  eggs  at  a  time  in  cracks  and  in  ten  days 'they  hatch  out 
into  larvae  which  pass  insensibly  into  adults  by  a  series  of  five  moultings;  this  deposit- 
ing of  eggs  occurs  about  four  times  a  year. 

The  bedbug  is  very  probably  the  intermediate  host  in  kala-azar  and  it  has  been 
incriminated  in  connection  with  typhus  fever  and  relapsing  fever.  It  can  also 
transmit  plague. 

A.  rotundata. — In  India  the  A.  rotundata  is  the  one  encountered.  It  is  of  a  dark 
mahogany  color,  has  a  smaller  head,  narrower  abdomen,  thick  rounded  prothoracic 
borders  and  is  more  densely  covered  with  hairs  than  A.  leclularia.  The  prothorax  of 
A .  leclularia  is  flattened  at  the  side. 

Reduviidae 

These  bugs  have  a  long  narrow  head  and  a  distinct  neck.  The 
antennae  are  long  and  slender.  The  antennae  in  the  genus  Conorhinus 
are  inserted  about  midway  between  the  eyes  and  point  of  the  head. 

Conorhinus  sanguisuga. — This  is  known  as  the  Texas  or  Mexican  bedbug,  and 
was  formerly  the  foe  of  the  common  bedbug,  but  having  gotten  a  taste  for  human 
blood  through  the  Cimex  or  Acanthia,  it  now  prefers  man.  It  is  extending  toward 
the  North.  It  has  wings.  The  bites  are  much  more  severe  than  those  of  the 
common  bedbug.  It  is  of  a  dark  brown  color,  nearly  an  inch  in  length,  with  a  long, 
flat,  narrow  head  and  a  short  thick  rostrum.  They  can  run  as  well  as  fly.  They 
bite  at  night. 

Conorhinus  megistus. — This  is  called  "Barbeiro"  in  Brazil  on  account  of  its 
preference  for  biting  the  face.  The  Schizotrypanum  cruzi  undergoes  a  develop- 
mental  cycle  in  this  bug  which  transmits  the  disease. 

SlPHONAPTERA 

These  are  laterally  flattened,  markedly  chitinized,  wingless  insects 
which  undergo  a  complete  metamorphosis. 

Pulicidse 

This  family  is  divided  into  two  subfamilies — the  Pulicinae  and  the 
Sarcopsyllinae.  In  the  former  the  female  remains  practically  un- 
changed with  freedom  of  movement  after  fecundation,  while  in  the 
latter  the  abdomen  becomes  enormously  distended  with  eggs,  and  the 
female  remains  fixed  in  the  burrow  which  she  has  made  under  the  skin. 

Pulcinse. — Formerly,  with  the  exception  of  infection  with  Dipylidium  caninum, 
the  fleas  were  only  under  suspicion  as  carriers  of  disease;  ideas  having  been  enter- 
tained as  to  their  being  possible  transmitters  of  relapsing  fever,  typhus  fever  and 
kala-azar.  Trypanosoma  lewisi  is  transmitted  by  fleas,  either  Pulex  irritans  or 
C.  cams.  The  trypanosome  undergoes  development  in  the  flea  and  the  infecting 


FLEAS  349 

material  is  in  the  fasces  of  the  flea  and  transmission  occurs  by  the  licking  on  the  part 
of  the  rat  of  faeces  from  an  infected  flea.  The  infection  has  no  connection  with  the 
puncture  wound  of  the  flea  as  is  the  case  with  plague.  As  a  result  of  the  convincing 
experiments  of  the  Indian  Plague  Commission,  their  role  in  the  transmission  of 
plague  has  been  absolutely  established.  It  is  by  the  bite  of  the  Xenopsylla  cheopis 
that  plague  is  chiefly  transmitted  from  rat  to  rat,  and  in  bubonic  and  septicaemic 
plague  it  is  apparently  the  intermediary  in  human  infection. 

The  average  capacity  of  a  flea's  stomach  is  about  0.5  cu.  mm.  so  that  with  a  rat 
dying  with  septicaemic  plague  and  with  possibly  100,000,000  bacilli  to  i  c.c.  of 
blood  the  flea  would  take  in  about  5000  bacilli.  Furthermore  these  multiply  in 
the  alimentary  canal  so  that  the  digested  blood  teems  with  bacilli  when  reaching 
the  anus  of  the  flea.  The  plague  bacilli  are  passed  out  with  the  fasces  and  these 
being  rubbed  into  the  puncture  of  the  flea  bite^  bring  about  infection.  Regurgitation 
as  result  of  obstruction  by  masses  of  plague  bacilli  in  the  oesophagus  causes  in- 
jection of  plague  bacilli  into  rat  or  man  in  the  act  of  biting.  This  is  more  important 
than  the  faeces  inoculation  method.  The  puncturing  apparatus  of  the  flea  consists 
of  a  pointed  epipharynx  and  two  distally  serrated  mandibles.  These  chitinous 
biting  parts  are  contained  in  the  labium  which  divides  distally  into  two  labial  palps. 
The  maxillae  are  conspicuous  triangular  structures  and,  projecting  farthest  anteriorly, 
are  the  conspicuous  four-jointed  maxillary  palps,  often  mistaken  for  antennae. 
By  the  apposition  of  the  internally  grooved  mandibles  to  the  epipharynx  a  tube  is 
formed  through  which  the  blood  is  sucked  up.  The  antennas  are  inconspicuous  and 
are  in  close  apposition  to  the  sides  of  the  head,  behind  the  eyes,  and  can  only  be 
well  made  out  with  a  lens.  Fleas  have  three  pairs  of  legs,  and  the  male  can  be 
distinguished  from  the  female  by  its  smaller  size  and  the  conspicuous  coiled-up 
penis  within  the  abdomen.  The  female  has  a  conspicuous  gourd-like  spermatheca 
which  varies  in  shape  in  different  species.  A  very  prominent  structure  is  a  pitted 
plate  in  the  ninth  abdominal  segment  (pygidium).  Of  importance  in  classification 
are  prominent  bristles  originating  from  the  seventh  abdominal  segments  and 
projecting  over  the  pygidium.  These  bristles  vary  in  number  and  are  known 
as  antepygidial  bristles. 

The  body  of  the  flea  is  flattened  laterally.  They  may  or  may  not  have  eyes,  and 
certain  conspicuous  structures  called  combs  are  of  importance  in  classification.  In 
the  metamorphosis  of  the  flea  the  eggs  are  hatched  out  in  dust  of  crevices,  etc.,  into 
bristled  larvse  in  about  one  week.  The  larva  forms  a  cocoon  and  develops  into  a 
nymph  which  has  three  pairs  of  legs.  The  ftymphs  emerge  from  the  cocoon  as  adult 
fleas  in  about  three  weeks  after  the  larva  forms  it. 

KEY  TO  THE  FLEAS 
A.  With  combs, 
i.  Eyes  present. 

(a)  Combs  along  inferior  border  of  head  and  on  prothorax. 
Ctenocephalus  serraticeps. 

(b)  Combs  only  on  prothorax.     Ceratophyllus  fasciatus  (with 
only  one  antepygidial  bristle  on  each  side). 
Hoplopsyllus  anomalus  (with  many  antepygidial  bristles). 


350 


THE   INSECTS 


2.  Eyes  absent. 

(a)  Collar  of  combs  on  prothorax  and  four  short  ones  along 

inferior  border  of  head.     Ctenopsylla  musculi. 
B.  Without  combs. 

(a)  Ocular  bristle  arises  near  upper  anterior  margin  of  eye.  A 
line  between  this  and  the  oral  bristle  approximately  ver- 
tical. Two  bristles  posterior  to  antennae.  Xenopsylla 
cheopis.  Loemopsylla  cheopis.  Formerly  Pulex  cheopis. 


-ZArery- 


FIG.  84. — i  and  2,  male  and  female  Xenopsylla  cheopis.     3,  Head  of  Ceratophyllus. 
4  and  5,  male  and  egg  distended  female  of  Sarcopsylla  penetrans. 

(b)  Ocular  bristle  arises  near  lower  anterior  margin  of  eye.  A 
line  between  this  and  the  oral  bristle  approximately  hori- 
zontal. One  bristle  posterior  to  antennae.  Pulex  irritans. 

The  common  human  flea  of  Europe  is  the  Pulex  irritans;  that  of  the  United  States 
the  Ctenocephalus  serraticeps  or  dog  flea.  The  flea  that  is  prominently  implicated 
with  plague  is  the  Xenopsylla  cheopis,  on  account  of  its  being  the  common  rat  flea 
of  India,  where  it  has  been  much  studied.  It  resembles  P.  irritans  t  but  is  more  yellow 
than  brown  in  color.  It  also  has  a  greater  number  of  bristles  on  the  head.  The 
ocular  bristle  runs  above  and  in  front  of  the  eye;  that  of  P.  irritans  below.  It  is 
principally  the  flea  of  Mus  decumanus  (M.  norvegicus),  the  sewer  rat;  but  the  house 
rat,  M .  rattus,  becomes  infected  from  coming  in  contact  with  the  sewer  rat  in  the 


THE  CHIGOE  351 

basement.  In  the  U.  S.  the  ground  squirrel,  Citellus  beechyi  acts  as  a  reservoir  of 
plague  and  has  as  its  flea  Hoplopsyllus  anomalus. 

Ceratophyllus  fasciatus  is  the  common  rat  flea  of  Europe  and  the  U.  S.  In  the 
tropics  X.  cheopis  is  the  common  rat  flea  (98%  in  India).  Ctenocephalus  serrati- 
ceps,  Ctenopsylla  musculi  and  Pulex  irritans  have  also  been  frequently  found  on 
both  Mus  norvegicus  and  M.  rattus.  To  distinguish  M .  norvegicus  from  M.  rattus 
we  have  in  the  former  (i)  ears  which  barely  reach  the  eyes  when  laid  forward  and  (2) 
tail  rather  shorter  than  length  of  head  and  body  together  (only  89%  of  length  of  head 
and  body  together).  With  M.  rattus  the  tail  is  longer  than  the  head  and  body 
together  (25%  longer)  and  the  extended  ear  covers  or  reaches  beyond  the  middle  of 
the  eye.  M.  rattus  has  a  sharper  nose,  longer  and  more  delicate  tail  and  thinner  ears 
than  M.  norvegicus  (formerly  M.  decumanus). 

M.  alexandrinus  is  a  variety  of  M.  rattus.  Rats  and  mice  belong  to  the  family 
Muridae  and  the  common  mouse  is  M.  musculus.  They  belong  to  the  order  of 
Rodentia  of  the  class  Mammalia. 

Sarcopsyllinae 

Belonging  to  the  subfamily  Sarcopsyllinae,  the  Sarcopsylla  penetrans  (Derma- 
tophilus  penetrans}  is  of  great  importance  in  tropical  countries.  It  is  known  as 
the  chigoe,  nigua,  or  jigger.  The  male  and  virgin  female  are  unimportant  as  they 
do  not  penetrate  the  skin  but  act  as  ordinary  fleas.  The  female,  which  when  un- 
impregnated  is  only  about  ^4  inch  long,  when  impregnated  bores  its  way  into 
the  skin  of  man,  especially  about  the  toes,  soles  of  the  feet  or  finger-nails,  and  in  the 
chosen  site  develops  enormously,  becoming  as  large  as  a  small  pea.  This  enlarge- 
ment takes  place  in  the  second  and  third  abdominal  segments  and  is  packed  with  eggs 
measuring  about  400  microns  long  and  numbering  about  100.  A  small  black  spot  in 
the  center  of  a  tense  rather  pale  area  is  characteristic.  The  metamorphosis  is 
similar  to  that  of  the  flea.  Sarcopsylla  can  be  differentiated  from  the  flea  by  the  pro- 
portionately larger  head  to  the  body,  and  especially  by  the  fact  that  the  head  is  the 
shape  of  the  head  of  a  fish,  distinctly  pointed.  With  the  fleas  the  lower  border  of  the 
head  comes  out  in  a  straight  line  to  join  the  curve  of  the  upper  part.  In  the  Sarco- 
psylla lower  and  upper  border  of  head  are  both  curved. 

DlPTERA 

The  insects  of  the  order  Diptera  are  of  great  importance  medically 
in  a  variety  of  ways,  either  by  the  direct  irritation  of  their  bites,  by  their 
transmitting  disease  directly,  as  does  the  common  house  fly  typhoid 
fever,  or  by  acting  as  intermediate  or  definitive  hosts  for  various 
parasites.  They  are  characterized  by  mouth  parts  formed  for  punctur- 
ing, sucking,  or  licking.  They  present  a  complete  metamorphosis,  larva, 
pupa,  and  imago.  As  a  rule,  the  Diptera  have  a  distinct  pair  of  wings 
the  second  pair  being  rudimentary. 

The  order  Diptera  is  usually  divided  into  the  following  suborders:  i.  Orthorrha- 
pha:  Diptera  with  larvae  having  a  differentiated  head.  The  imago  breaks  through 


352 


THE   INSECTS 


al  space 


the  larval  or  pupal  case  by  a  T-shaped  break  and  has  no  frontal  lunule  (an  oval  sj 
just  above  the  root  of  the  antennae).     The  Orthorrhapha  are  divided  into:  a.  Nemo- 
cera  (with  long,  many  jointed  antennae)  and  b.  Brachycera  (with  short  antennae). 

The  Nemocera  are  generally  midge-like  insects  and  have,  as  a  rule,  long  slender  palps 
(mosquito).  The  Brachycera,  however,  are  seldom  midge-like.  The  antennae  are 
composed  of  only  two  or  three  simple  joints  with  or  without  style  or  arista.  The  palps 
are  almost  always  short  and  never  more  than  two- jointed.  2.  Cydorrhapha:  larvae 
without  differentiated  head.  The  imago  escapes  through  an  anterior  opening  and  has 
a  lunule  and  ptilinum  (an  inflatable  projecting  organ  just  above  the  root  of  the  an- 
tennae). If  the  halteres  are  covered  by  a  scale  (squama)  we  have  calyptrate  Cyclor- 
rhapha;  if  not,  acalyptrate.  These  squamae  are  large  enough  in  the  calpytrate  species 
to  even  conceal  the  halteres  when  the  fly  is  looked  at  from  above.  3.  Pupipara:  the 
larvae  are  extruded  from  the  mother  and  almost  immediately  begin  the  pupal  life. 
Leathery  flies  with  poorly  developed  wings  (Hippobocidae). 

The  males  of  flies  where  the  two  compound  eyes  come  together  above  the  antennae 
are  referred  to  as  holoptic,  if  more  or  less  widely  separated  as  dichoptic.  Ocelli  are 
three  single  eyes  usually,  when  present,  situated  in  the  triangular  space  between  the 
compound  eyes  in  the  front  (the  space  separating  the  compound  eyes). 

The  anterior  portion  of  the  head  which  lies  below  the  origin  of  the 
antennae  is  the  face  and  on  each  side  of  the  face  we  have  the  cheeks 
which  should  be  studied  as  to  presence  of  abundance  of  hairs.  The 
antennae  which  separate  the  front  from  the  face  are  of  great  importance 
in  classification.  In  the  Muscidae  the  appearance  of  a  feathery  struc- 
ture, projecting  from  the  terminal  segment  of  the  antennae,  and  called 
the  arista,  is  important.  This  may  be  bare  or  feathered  and  the 
feathering  may  be  only  on  one  side  or  of  one  part. 

In  studying  the  biting  flies  it  is  very  important  to  recognize  the  anterior,  small, 
or  mid-cross  vein.  This  short  transverse  rib  or  vein  is  the  key  to  wing  venation. 
Beneath  it  is  the  discal  cell  and  it  bounds  the  first  posterior  cell  internally  or  basally. 
The  fourth  longitudinal  vein,  which  touches  the  bottom  of  the  mid-cross  vein,  is  of 
particular  importance  as  it  gives  different  shapes  to  the  first  posterior  cell  as  it  runs 
along  the  lower  border  of  this  cell.  The  closed-in  discal  cell  is  below  the  fourth 
longitudinal  vein.  The  character  of  the  antennae  should  also  be  noted  carefully. 
The  study  of  the  bristles  about  head,  thorax,  and  abdomen  (chaetotaxy)  is  more 
difficult.  Anyone  taking  up  the  study  of  flies  should  carefully  note  the  wings,  etc., 
of  Musca  domestica.  By  putting  a  few  house  flies  on  moist  horse  manure  in  a  gauze- 
covered  bottle  the  entire  metamorphosis  may  be  observed. 

Tabanidae 

This  is  the  family  of  horseflies,  gadflies,  breeze  flies  or  green-headed  flies.  It 
is  the  most  numerous  family  of  the  Diptera — there  being  more  than  1000  species. 
The  females  are  blood  suckers;  the  males  live  on  flowers  and  plant  juices.  The 
eyes  are  usually  very  brilliant  in  color,  and  in  the  male  make  up  the  greater  part  of 
the  head. 


THE  HOUSEFLY 


353 


They  belong  to  the  suborder  Orthorrhapha  and  in  the  group  of  short  antennae  flies 
(Brachycera).  Five  posterior  cells  are  always  present. 

The  antennae  consist  of  three  segments.  No  arista.  The  epipharynx  is  tube 
like,  the  hypopharynx  has  a  groove  and  both  are  awl-shaped.  The  pair  of  maxillae 
are  serrated  and  the  mandibles  lancet  like.  They  have  rather  coarse  maxillary 
palps.  The  labellae  are  prominent  at  the  extremity  of  the  fleshy  labium.  They  are 
thick  set  flies  and  rarely  show  color.  The  body  of  the  larva  has  eleven  segments 
with  a  small  but  distinct  head.  The  eggs  are  deposited  in  masses  on  the  leaves  or 
stems  of  plants  about  marshy  places.  The  larva  is  carnivorous. 

Tabanus  autumnalis. — Is  about  %  inch  long;  it  is  dark  in  color,  and  has  four 
longitudinal  bands  on  the  thorax.  The  last  joint  of  the  antennae  has  a  crescentic 
notch.  The  wings  do  not  overlap. 

Haematopota  pluvialis. — In  the  Hosmatopota  there  is  no  crescentic  antennal  notch, 
and  the  wings  overlap.  The  abdomen  is  narrower  than  in  Tabanus.  The  brimp,  one 
of  the  Hcematopota,  bites  man  severely. 


FIG.  85. — Common  housefly  (Musca  domestica):  Puparium  at  left;  adult  next, 
larva  and  enlarged  parts  at  right.  All  enlarged.  From  circular  71  (by  L.  O. 
Howard),  Bureau  of  Entomology,  U.  S.  Department  of  Agriculture. 

Pangonia  beckeri. — The  genus  Pangonia  is  characterized  by  a  very  long,  slender, 
and  more  or  less  horizontal  proboscis. 

Chrysops  dispar. — Chrysops  has  three  ocelli,  in  this  respect  differing  from  the 
genera  Tabanus  and  Hcematopota.  The  wings  are  widely  separated  and  spotted. 
The  antennas  of  Chrysops  are  especially  long  and  slender.  Chrysops  and  Hcemato- 
pota produce  the  greatest  amount  of  pain  from  their  bites.  The  Tabanidoe  except 
Chrysops  are  not  implicated  as  intermediate  hosts  in  the  transmission  of  disease.  By 
their  bites,  however,  they  may  transmit  disease  directly,  as  with  anthrax.  Two 
species  of  Chrysops,  C.  dimidiata  and  C.  silacea,  have  been  found  to  transmit 

Filaria  loa. 

Muscidae 

The  Muscidce,  Sarcophagidae,  and  (Estridae  are  calyptrate  Cyclorrhapha. 
Musca  domestica. — The  common  housefly,  Musca  domestica,  is  the  best  example  of 
this  family. 
23 


354 


THE   INSECTS 


fourth 


The  arista  is  feathered  both  dorsally  and  ventrally  with  straight  hairs.  The  fourtl 
longitudinal  vein  bends  down  in  a  rather  sharp  angle  as  compared  with  Stomoxys, 
which  gives  the  first  posterior  cell  of  the  latter  rather  a  fusiform  appearance.  The 
eyes  are  close  together  in  the  male,  far  apart  in  the  female.  The  female  lays  about 
125  eggs  in  a  heap  preferably  in  fermenting  horse  manure.  The  larva  comes  out  in 
about  thirty-six  hours.  Very  characteristic  are  the  stigmata  decorating  the  blunt 
posterior  ends.  (See  illustration.) 

The  larval  stage  lasts  seven  to  ten  days  and  then  the  barrel-shaped  pupal  stage 


Phlebotomus     [Psychodidae] 


(Phoridae) 

FIG.  86. — Wing  venation  of  Diptera.     A,  First  posterior  cell;  £,  discal  cell;  b, 
mid  cross- vein;  a,  auxiliary  vein;  C,  marginal  cell;  D,  submarginal  cell. 


is  entered  upon.  This  lasts  about  three  days  when  the  adult  fly  emerges.  This  is 
termed  a  "coarctate"  pupa.  The  larva  shrinks  and  is  surrounded  by  its  old  skin 
which  is  termed  a  puparium.  This  fly  is  incapable  of  biting,  the  piercing  organs 
being  fused  with  the  labium,  but  may  transmit  disease  directly,  carrying  infectious 
material  from  the  source,  as  in  faeces,  to  the  food  about  to  be  ingested.  Their  r6le  in 
typhoid  fever  is  one  of  immense  importance.  By  reason  of  its  hairy  sticky  legs, 
habits  of  frequent  defecation  and  constant  regurgitation  the  housefly  is  an  important 


TSETSE  FLIES 


355 


agent  in  the  spread  of  cholera,  dysentery,  infantile  diarrhoeas  and  tropical  ophthal- 
mias as  well  as  typhoid. 

In  the  Muscidae  the  antennae  hang  down  in  front  of  the  head  in  three  segments 
and  have  an  arista  plumose  to  the  tip.  The  first  posterior  cell  is  narrowed.  There 
are  no  bristles  on  abdomen  except  at  tip. 

(I)  Stomoxys,  Hazmatobia  and  Glossina  have  a  more  or  less  elongated 
proboscis  adapted  for  biting.     Stomoxys  has  delicate  palpi,  shorter  than 
the  proboscis,  and  arista  feathered  only  on  the  dorsal  side  with  straight 
hairs.     Hasmatobia  has  club-like  palpi  about  as  long  as  proboscis  and 
arista  with  hairs  dorsally  and  ventraUy.    Glossina  has  thick  set  but 
not  clubbed  palpi  and  an  arista  feathered  on  the  dorsal  side  with 
branching  hairs. 

(II)  Musca,    Callipkora,    Chrysomyia,  Lucilia,   and  CordyloMa  do 
not  have  a  proboscis  adapted  for  biting. 

Stomoxys  calcitrans. — These  greatly  resemble  the  common  housefly  in  size  and 
shape.  They  can  be  easily  distinguished  by  the  black,  piercing  proboscis  extend- 
ing beyond  the  head.  There  are  longitudinal  stripes  on  the  thorax  and  spots  on  the 
abdomen.  The  proboscis  on  examination  will  be  seen  to  be  bent  at  an  angle  near  its 
base.  The  palps  are  short  and  slender.  The  wings  diverge  widely. 

The  female  lays  about  60  banana-shaped  eggs  in  horse  manure.  These  hatch 
out  in  three  days  as  larvae  which  turn  into  pupae  in  two  or  three  weeks.  After  about 
ten  days  the  fly  emerges.  The  genus  Stomoxys  includes  vicious  biters.  This  is  the 
fly  which  comes  into  houses  before  a  rain,  and  which  has  given  the  common  housefly 
the  reputation  of  biting  before  a  rain.  Stomoxys  may  be  implicated  in  transmitting 
surra  (Trypanosoma  evansi). 

It  assumed  great  importance  as  a  transmitter  of  poliomyelitis  and 
possibly  of  pellagra  a  few  years  ago — views  now  discredited. 

The  horsefly  (H&matobia  irritans)  rarely  bites  man.  In  these  the 
palpi  are  much  longer  than  in  Stomoxys,  being  as  long  as  proboscis. 
These  palps  are  also  thick  and  spatulate. 

Glossina  palpalis. — This  is  the  tsetse  fly  that  is  responsible  for  the 
transmission  of  human  trypanosomiasis  (sleeping  sickness). 

The  tsetse  fly  is  a  small  brownish  fly  about  H  inch  long.  The  proboscis 
extends  vertically  and  has  a  bulb  at  its  base.  The  arista  is  plumose  only  on  the 
upper  side  and  the  individual  hairs  are  themselves  feathered.  The  wings  are  carried 
flat,  closed  over  one  another  like  the  blades  of  a  pair  of  scissors  and  project  beyond 
the  abdomen.  The  most  characteristic  feature  of  the  tsetse  fly  is  the  way  the  fourth 
longitudinal  vein  bends  up  abruptly  to  meet  the  midcross  vein  and  then  curves 
downward  to  run  parallel  with  the  third  longitudinal  vein.  In  Stomoxys,  the  wings 
separate;  in  Hcematopota  they  just  meet,  and  in  Glossina  they  cross.  Glossinae  bite 
chiefly  in  the  daytime. 

The  tsetse  fly  is  much  like  Stomoxys,  but  has  a  branching  of  the  feathering  of  the 


356 


THE    INSECTS 


arista,  long  palps,  a  bulb  to  the  proboscis  and  a  characteristic  upbending  of  the 
fourth  longitudinal  vein  to  meet  the  midcross  vein.  The  female  deposits  her  larva 
near  a  shady  place  upon  loose,  dry,  sandy  soil.  Moisture  and  sunlight  are  not  favor- 
able for  pupal  development,  the  sun  being  particularly  injurious,  so  that  pupae, 
buried  only  an  inch  deep  and  away  from  shade,  are  killed.  This  fact  has  been 
utilized  in  prophylaxis  by  cutting  down  the  trees.  The  trouble  is  that  the  bush 
growth  which  soon  follows  is  favorable  as  shade  for  the  pupae. 

The  female  gives  birth  to  a  single,  yellowish  brown,  motile  larva,  which  is  almost  as 
large  as  the  mother  and  which,  upon  reaching  the  ground,  bores  its  way  into  a  coarse, 
sandy  soil  for  a  depth  of  about  2  inches  and  then  becomes  a  pupa.  The  larval  stage 


FIG.  87. — Insects  in  which  the  adult  stage  is  important,  (i)  Stomoxys  calcitrans; 
(2)  S.  calcitrans,  larva;  (3)  Tabanus  bovinus;  (4)  Tabanus  larva;  (5)  Glossina  palpalis; 
(6)  G.  palpalis,  side  view;  (7)  G.  palpalis  pupa;  (8)  Glossina  palps  and  arista. 

in  the  mother  lasts  about  two  weeks  and  the  pupal  stage  in  the  ground  about  a 
month, 

Male  and  female  flies  bite  and  transmit  the  disease.  They  bite  in  the  daytime, 
usually  from  9  A.M.  to  4  P.M.,  and  will  bite  in  the  sunlight. 

With  a  view  to  eradication  of  the  disease  certain  areas  have  been  depopulated,  but 
upon  examining  the  flies  caught  in  the  district  a  year  or  more  later,  infected  flies 
have  been  obtained.  This  would  indicate  some  other  reservoir  than  man.  It  is 
now  generally  conceded  that  the  trypanosome  strain  in  the  antelope  is  the  same  as 
T.  rhodesiense,  both  being  transmitted  by  G.  morsitans.  Taute  however  believes  them 
different  as  he  not  only  injected  blood  containing  such  trypanosomes  into  himself, 


FLESH  FLIES  257 

with  negative  result,  but  also  allowed  flies  which  had  fed  on  antelopes,  which  were 
infective  for  laboratory  animals,  to  feed  on  himself,  likewise  with  negative  result.  It 
is  a  well-known  fact  that  men  in  good  condition  are  refractory  to  trypanosome  infec- 
tion so  that  this  courageous  experiment  does  not  prove  the  antelope  strain  to  be 
different  from  the  human  one. 

One  measure  that  has  been  proposed  is  to  kill  off  the  big  game  from  a  certain  area 
with  a  view  to  depriving  the  flies  of  their  main  source  of  infection. 

The  probabilities  of  an  animal  reservoir  for  T.  gambiense  however  is  not  so  well 
settled.  ^Many  think  that  we  may  have  trypanosome  carriers  and  that  such  persons 
in  the  enjoyment  of  health  may  act  as  reservoirs  of  the  virus.  Koch  suggested  that 
crocodiles  were  important  factors  in  the  life  of  the  tsetse  flies  and  recommended 
the  destruction  of  the  crocodile  eggs. 

Glossina  morsitans  transmits  the  cattle  trypanosome  disease,  nagana  and  the 
human  infection  due  to  Trypanosoma  rhodesiense. 

Auchmeromyia  luteola.— This  is  an  African  fly,  the  larva  of  which  is  known  as 
the  "Congo  floor  maggot,"  and  is  a  blood  sucker.  The  larva  is  of  a  dirty-white 
color  and  about  %j  inch  long.  It  crawls  out  at  night  and  feeds  on  the  sleeping  native. 
This  is  the  only  known  instance  of  a  blood-sucking  larva. 

Calliphora  vomitoria  and  Lucilia  caesar.— These  are  flies  with  brilliant  metallic- 
colored  abdomens,  commonly  called  blow  flies  in  the  case  of  Calliphora  and  blue- 
bottle flies  for  Lucilia.  They  deposit  their  eggs  on  tainted  meat  and  in  wounds. 
Many  cases  of  obscure  abdominal  trouble  are  probably  due  to  the  larvae  of  these 
flies.  Intestinal  myiasis  is  undoubtedly  of  greater  importance  than  has  been 
thought.  The  larvae,  with  hook-like  projections  anteriorly  and  a  ringed  body,  can 
easily  be  recognized  in  the  faeces.  They  have  been  mistaken  for  flukes.  They 
also  have  a  tendency  to  be  attracted  by  those  with  ozena  and  the  larvae  may  develop 
in  the  nostrils.  Cheeks  bare  in  Lucilia,  hairy  in  Calliphora. 

Chrysomyia  macellaria.— This  is  known  as  the  screw- worm  when  in  the  larval 
stage.  The  adult  fly  resembles  the  blue-bottle  flies.  It  is  distinguished  from  them, 
however,  by  the  presence  of  black  stripes  on  thorax.  These  flies  are  very  common 
over  nearly  all  North  and  South  America.  The  eggs  which  number  250  or  more, 
when  deposited  in  the  nostrils  or  in  wounds,  develop  into  the  screw-worm  larva, 
which  may,  by  going  up  into  the  frontal  sinus,  cause  death.  These  larvae  have 
twelve  segments  with  rings  of  minute  spines. 

Ochromyia  anthropophaga  (Cordylobia  anthropophaga  or  Tumbu  Fly). — This 
is  an  African  fly  whose  larvae  develop  under  the  skin  of  man  and  animals.  It  is 
known  as  the  Ver  de  Cayor.  The  larva  resembles  the  Ver  Macaque,  is  rather 
barrel-shaped  and  beset  with  small  spines.  It  bores  its  way  into  the  skin  and  makes 
a  lesion  like  a  boil  which  has  a  central  opening  through  which  the  larva  breathes. 

Sarcophagidae 

These  are  known  as  "flesh  flies."  The  most  important  characteristic  is  the  fact 
that  the  arista  is  plumose  up  to  the  mid-point,  beyond  which  it  is  bare.  They  are 
usually  thick  set  and  moderately  large  flies. 

Sarcophaga  carnaria. — This  is  a  grayish  fly  with  three  stripes  on  thorax  and 
black  spots  on  each  segment  of  the  abdomen.  It  is  viviparous.  The  larvae  gain 
access  to  nasal  and  other  cavities  and  there  develop.  Cases  of  death  have  been 


358 


THE   INSECTS 


of  war 


reported.  Naturally,  the  fly  deposits  its  larvae  on  decaying  flesh.  In  times  of 
all  of  these  flies  become  important  by  reason  of  "maggots"  in  the  wound.  These 
larvse  are  the  most  common  ones  in  intestinal  myiases.  The  mouth  booklets  are 
strongly  curved  and  separate.  Each  abdominal  segment  has  a  girdle  of  spines. 
The  anterior  end  is  somewhat  pointed.  The  hind  stigmal  plate  is  in  a  deep  cavity. 


CEstridse 

The  flies  of  this  family  are  usually  called  botflies.  The  mouth  parts  are  almost 
vestigial.  They  have  a  large  head  with  a  somewhat  bloated-looking  lower  portion. 
They  are  often  rather  hairy.  The  larvae  which  develop  from  the  eggs  are  parasitic 
either  in  the  alimentary  canal  or  the  subcutaneous  tissues. 


FIG.  88. — Insects  in  which  the  larval  stage  is  important,  (i)  Chrysomyia  macel- 
laria;  (2)  C.  larva;  (3)  Dermatobia  cyaniventris  larva,  early  stage  (ver  macaque); 
(4)  D.  cyaniventris  larva,  later  stage  (torcel  or  berne);  (5)  D.  cyaniventris;  (6) 
Auchmeromyia luteola;  (7)  A.luteola,  larva;  (8)  Sarcophaga magnifica;  (9)  S.  magnifica 
larva;  (10)  Anthomyia  pluvialis;  (n)  A.  phtvialis  larva. 

Dermatobia  cyaniventris. — These  are  large,  thick-set  flies  about  %  inch  long, 
with  prominent  head  and  eyes,  small  antennae,  and  a  marked  narrowing  at  the 
junction  of  thorax  and  abdomen.  The  thorax  is  grayish  and  the  abdomen  a  metallic 
blue.  The  larvae  are  deposited  under  the  skin  in  various  parts  of  the  body.  When 
the  larvae  move  they  cause  considerable  pain.  At  first  the  larva  is  club-shaped, 
but  later  on  it  becomes  oval.  The  former  is  called  Ver  Macaque,  the  latter  Torcel. 

Hypoderma  diana.— The  larval  form  of  this  fly  has  been  reported  three  times 


THE  SCREW-WORM  359 

for  man.    It  forms  tumors  under  the  skin  which  it  is  thought  may  reach  this  location 
by  proceeding  in  some  way  from  the  alimentary  canal. 

In  Hypoderma  the  arista  is  bare  while  in  Dermatobia  the  upper  border  is  plumose. 

MYIASES 

Ver  Macaque. — The  best  known  of  these  myiases  is  that  due  to  the 
larva  of  a  gadfly,  Dermatobia  cyaniventris. 

The  larva  is  at  first  club-shaped  and  in  this  stage  is  called  ver  macaque.  Later  on 
it  becomes  worm-shaped  and  is  then  called  torcel  in  Venezuela  or  berne  in  Brazil. 
The  natives  of  most  of  the  countries  where  the  infection  is  found  have  called  the 
larvae  "mosquito  worms"  or  "gusano  de  zancudo"  and  they  have  even  incriminated 
large  mosquitoes  belonging  to  the  genus  Psorophora  as  being  responsible  for  the 
infections. 

Surcouf  has  noted  that  these  fly  larvae  have  been  found  cemented  to  mosquitoes 
of  the  genus  Janthinosoma  by  a  glue-like  substance.  These  mosquitoes  are  vicious 
biters  and  evidently  the  young  larvae  escape  from  the  eggs  attached  to  the  mosquito 
and  enter  the  wound  made  by  the  biting  parts  of  the  mosquito.  Some  have  thought 
that  D.  cyaniventris  deposits  its  eggs  in  a  glue-like  material  on  the  leaves  of  plants 
and  that  they  stick  to  mosquitoes  flying  about  such  plants.  From  the  facts  that 
these  eggs  apparently  only  become  attached  to  this  particular  mosquito  and  further 
in  that  the  eggs  are  attached  in  a  constant  manner  with  the  hatching  end  outward  it 
would  seem  that  the  mother  fly  must  in  some  way  seize  the  mosquito  and  deposit  her 
eggs  on  it.  As  the  larva  grows  in  the  subcutaneous  tissues  of  man  or  other  animals 
a  tumor-like  swelling  develops  with  a  central  orifice,  toward  which  the  posterior 
extremity  of  the  larva  points  and  through  which  it  takes  air  into  its  spiracles. 

The  swelling  somewhat  resembles  a  blind  boil  and  may  be  as  large  as  a  pigeon's 
egg- 

These  gadfly  boils  tend  to  break  down  and  discharge  a  sero-purulent  fluid  and  it  is 
supposed  that  the  larva,  when  mature,  escapes  as  a  result  of  the  disintegration  of  the 
tumor. 

In  Brazil  they  make  tobacco  juice  applications  which  cause  the  larva  to  protrude 
and  then  squeeze  it  out.  The  injection  of  a  little  chloroform  into  the  larva  with  a 
hypodermic  syringe,  prior  to  its  extraction  with  a  forceps,  makes  the  process  less 
painful. 

The  Screw-worm. — This  is  the  larva  of  a  blue-bottle  fly,  Chrysomyia 
macellaria,  which  differs  from  the  common  blue-bottle  fly,  Lucilia, 
by  having  three  black  lines  on  scutum, 

This  muscid  fly  lays  200  to  300  eggs  in  wounds  or  orifices  having  offensive  dis- 
charges, as  from  nose,  ears,  etc.  The  larvae  burrow  into  the  adjacent  tissues  and 
cause  frightful  destruction  of  all  soft  parts.  The  mature  larvae  are  a  little  more 
than  %  inch  long  and  have  circlets  of  spines  around  each  of  the  1 2  segments. 

This  infection  is  especially  common  in  tropical  and  subtropical  America  and  is 
important  in  animals  as  well  as  man. 


THE   INSECTS 


Intestinal  Myiases 

In  the  tropics  vague  intestinal  disturbances  or  violent  abdominal 
cramping  may  be  brought  about  by  dipterous  larvae  in  the  intestinal 
canal.  The  symptoms  may  be  those  of  a  dysentery  and  may  be  at- 
tended with  fever  and  malaise. 

The  larvae  usually  obtain  access  to  the  alimentary  tract  in  food  taken  in  by  the 
mouth.  Flies  of  the  genus  Sarcophaga  are  prone  to  deposit  their  larvae  on  food, 
especially  meat  that  is  somewhat  tainted.  Other  flies,  as  Musca  or  Anthomyia, 


FIG.  89. — Markings  of  breathing  slits  on  posterior  stigmata  of  various  dipterous 
larvae,  i.  Musca  domestica,  showing  both  stigmata;  2.  Calliphora  vomitoria;  3. 
Stomoxys  calcitrans.  4.  Auchmeromyia  luteola;  5.  Cordylobia  anthropophaga;  6. 
Sarcophaga  magnifica. 

may  lay  their  eggs  on  food.     Flies  of  the  genus  Anthomyia  tend  to  lay  their  eggs  on 
plants. 

It  is  possible  for  a  fly  to  deposit  its  eggs  or  larvae  about  the  anus 
while  the  man  is  at  stool. 

Great  care  must  always  be  observed  to  assure  one's  self  that  fly  larvae,  which  may 
be  present  in  the  stool,  have  not  originated  from  larvae  deposited  on  the  stool  subse- 
quent to  its  passage. 

Aural  Myiases 

While  the  larva  of  Chrysomyia  macellaria,  known  as  the  "screw- 
worm,"  is  the  one  most  frequently  reported  from  the  external  auditory 


MYIASES  361 

canal  yet  many  such  cases  have  been  connected  with  the  larvae  of 
Sarcophaga  carnaria,  Calliphora  wmitoria  and  Anthomyia  pluvialis. 
These  larvae  are  usually  deposited  in  the  auditory  canals  of  those  with 
otorrhoea. 

The  symptoms  are  intense  earache,  giddiness  and  possibly  convulsions.  The 
larvae  tend  to  perforate  the  tympanic  membrane.  Instillations  of  10%  chloroform 
in  milk  or  the  use  of  oils  kill  the  larvse. 


DETERMINATION  OF  DIPTEROUS  LARV.E 

There  are  certain  points  in  the  anatomy  of  dipterous  larvae  which 
must  be  considered  in  recognition  of  the  genus  or  family  of  the  flies  con- 
cerned in  the  various  myiases.  The  broad  extremity  is  the  posterior 
one  and  the  tapering  one  the  anterior.  The  dark  hooklike  processes, 
which  may  be  in  pairs  or  fused,  project  from  the  anterior  or  head  end 
and  above  them  are  a  pair  of  projecting  papillae.  The  second  segment 
from  the  head  has  on  either  side  projecting  hand  or  fan-like  structures 
with  varying  numbers  of  terminal  divisions,  4  to  40  or  more.  These 
are  the  anterior  spiracles. 

The  large  terminal  segment  has  on  its  posterior  surface  two  chitinized  plates  with 
3  slits  of  various  architecture  in  each.  These  are  the  posterior  stigmal  plates  and 
are  the  structures  we  pay  particular  attention  to  in  identification.  In  the  early 
larval  stages  there  is  only  one  slit;  in  the  second  stage  there  are  two.  It  is  only  in 
the  fully  developed  larval  stage  that  we  note  the  characteristic  3  slit  stigmal  plates. 
The  presence  or  absence  of  a  rounded  protuberance  or  button  at  the  base  of  each 
stigmal  plate  should  be  looked  for.  The  area  carrying  the  stigmal  plates  may  be 
sunken  to  form  a  pit. 

KEY  TO  LARV2E  OF  THE  MYIASES   (BANKS) 

1.  Body  with  lateral  and  dorsal  spinose  processes Homalomyia. 

Body  without  such  processes 2 

2.  Body  ending  in  two  fleshy  processes;  rather  small  species 3 

Body  truncate  or  broadly  rounded  at  end 4 

3.  Processes  bearing  the  stigmal  plates;  body  about  5  mm.  long.  .  . .  Drosophila. 
Processes  not  bearing  the  stigmal  plates;  body  10  mm.  or  longer .  Piophila. 

4.  But   one  great   hook;    posterior  stigmal  plates   with   winding 

slits;  no  distinct  lateral  fusiform  areas;  tip  of  body  with 

few  if  any  conical  processes Muscinae. 

With  two  great  hooks;  slits  in  the  stigmal  plate  not  sinuous 5 

5.  No  tubercles  about  anal  area;  no  distinct  processes  around 

stigmal  field 6 

Distinct    tubercles   above   anal   area;   often   processes   around 

stigmal  field;  lateral  fusiform  areas  usually  distinct 7 


362  THE   INSECTS 

6.  Stigmal  plates  on  black  tubercles;  lateral  fusiform  areas  distinct.  Ortalidae 
Stigmal  plates  barely  if  at  all  elevated;  lateral  fusiform  areas 

indistinct;    stigmal  plates  often  contiguous  or  nearly  so; 

slits  long  and  subparallel Trypetidae. 

7.  Slits  in  stigmal  plates  rather  short,  and  arranged  radiately.  ...  8 
Slits  slender  and  subparallel  to  each  other 9 

8.  Two   tubercles   above  anal  area;   stigmal   field   with  distinct 

processes  around  it Anthomyiidae. 

Four  or  more  tubercles  above  anal  area;  slits  of  stigmal  plates 

usually  pointed  at  one  end Muscina. 

9.  A  button  to  each  stigmal  plate;  slits  rather  transverse  to  body. .  .    Calliphorinae. 
No  button  to  stigmal  plates,  slits  of  one  plate  subparallel  to 

those  in  opposite  plate;  plates  at  bottom  of  a  pit Sarcophagidae 


CHAPTER  XXI 
THE  MOSQUITOES 

MOSQUITOES  (Culicidae)  are  of  the  greatest  importance  medically, 
not  only  from  their  influence  upon  health  in  general  by  reason  of  inter- 
ference with  sleep  and  possibly  from  direct  transmission  of  disease,  but, 
more  specifically,  they  are  the  only  means  by  which  it  at  present  appears 
possible  to  bring  about  infection  with  such  diseases  as  yellow  fever, 
malaria,  filariasis,  and  possibly  dengue.  In  addition,  many  diseases 
of  animals  are  transmitted  by  mosquitoes. 

The  Culicidse  differ  from  all  other  Diptera  in  having  scales  on  their 
wings  and  generally  on  head,  thorax,  or  abdomen. 

To  identify  a  mosquito,  examine  a  wing  and  note  the  scales;  also  note  the  presence 
of  two  distinct  fork  cells  and,  in  addition,  that  the  costal  vein  passes  completely 
around  the  border  of  the  wing,  making  a  sort  of  fringe  with  its  scales.  Mosquitoes 
undergo  a  complete  metamorphosis,  there  developing  from  the  egg  a  voracious, 
rapidly  growing  larva;  next,  a  nongrowing,  nonf ceding  stage— the  pupa  or  nymph. 
In  this  latter  the  head  and  thorax  are  combined  is  an  oval  body,  from  the  back 
of  which  projects  the  siphon  tubes;  and  tucked  in  ventrally  is  a  small  tail-like 
appendage. 

The  fully  developed  insect  emerges  from  the  pupa. 

The  Culicidae  belong  to  the  suborder  Nematocera.  These  have  long  articulated 
antennas  and  include  four  families:  Culicidae,  Chironomidae,  Simulidae  and  Psycho- 
didaj. 

The  principal  mosquito-like,  blood-sucking  Diptera  which  are 
frequently  mistaken  for  mosquitoes— none  of  which  have  scales  on  their 
wings — are  the  following: 

1.  Chironomidae  or  Midges. — The  blood-sucking  species  of  Chironomidae,  which 
are  found  in-  most  parts  of  the  world,  belong  chiefly  to  the  genus  Ceratopogon.     These 
midges  are  of  very  small  size,  about  ^2  mch  long,  are  able  to  get  through  netting 
and,  usually  being  in  swarms,  they  are  exceedingly  troublesome.     The  antennae 
have  thirteen  joints  and  the  wings  are  shorter  than  the  abdomen  and  have  only 
longitudinal  veins.     One  of  the  midges,  the  "jejen"  of  Cuba,  is  a  great  scourge, 
its  small  size  enabling  it  to  enter  eyes  and  nostrils.     The  larva  of  Chironomus  is  a  red 
worm-like  creature;  the  pupa  has  a  tufted  head. 

2.  Simulidae  or  Buffalo  Gnats. — These  are  small  blood-thirsty  insects  only  about 
%  inch  in  length.     The  thorax  is  humped,  the  legs  are  short  and  the  proboscis 

363 


364 


THE   MOSQUITOES 


short  and  inconspicuous.  The  antennae  have  n  joints  but  are  rather  shoi 
One  species,  the  S.  damnosum,  known,  by  the  natives  of  Uganda  as  "Mbwa,"  is 
greatly  dreaded;  its  bites  causing  swellings  and  sores.  Sambon  has  considered 
Simulium  reptans  as  the  transmitting  agent  of  pellagra. 

3.  Psychodidse  or  Moth  Midges. — These  are  small,  hairy,  slender  midges,  with 
long  legs  and  a  short  proboscis.  The  antennae  are  long,  hairy  and  consist  of  12  to  1 6 
joints.  Palpi  four  jointed.  They  are  only  about  J^2  inch  in  length.  The  hairy 
wings  have  numerous  longitudinal  veins.  Some,  as  Phlebotomus,  have  an  enlongated 
proboscis  and  are  vicious  blood  suckers. 


FIG.  90. — Mosquito-like  insects  belonging  to  families  Chironomidae,  Simulidae 
and  Psychodidae.  (i)  Phlebotomus  papatasii;  (2)  P.  papatsii  (natural  size);  (3) 
P.  papatasii  (larva) ;  (4)  P.  papatasii  larva  (natural  size) ;  (5)  Ceratopogon  pulicaris; 
(6)  C.  pulicaris  (natural  size);  (7)  Chironomus  larva;  (8)  Attitude  of  a  Simulium; 
(9)  Simulium  reptans;  (10)  Larvae  of  Simulium. 

At  present,  of  the  genera  of  the  three  families  of  midges,  only  Phle- 
botomus is  known  to  transmit  disease.  P.  papatasii  transmits  phle- 
botomus  fever  in  the  Balkans.  P.  minutus  is  the  host  at  Aden. 
Another  species,  P.  perniciosus  can  transmit  the  disease.  These  moth 
midges  are  2  mm.  in  length  and  have  the  body  densely  covered  with 
long  yellow  hairs.  The  second  longitudinal  vein  has  three  distinct 
branches.  The  antennae  have  16  constricted  joints  and  the  proboscis 
is  as  long  as  the  head.  The  species  of  Phlebotomus  are  separated  by 


PHLEBOTOMUS  365 

slight  variations  in  wing  venation,  palpal  lengths,  etc.,  thus  the  second 
segment  of  palpi  of  P.  papatasii  is  a  little  longer  than  the  third  one 
while  with  P.  perniciosus  these  segments  are  of  equal  lengths.  In 
P.  minutus  the  second  segment  is  only  half  the  length  of  the  third. 
The  insect  lays  about  40  eggs  in  damp  dark  places.  The  period  of 
metamorphosis  from  egg  to  insect  is  about  one  or  two  months, 
according  to  temperature. 

Phlebotomus  larvae  die  out  in  dry  soil  and  very  wet  earth  is  un- 
favorable. Moderate  moisture  and  protection  from  light  seem  neces- 
sary for  their  development.  The  remains  of  dead  insects  also  seem 
to  make  good  breeding  places.  It  is  in  cracks  of  old  damp  brick 
or  stone  walls  that  the  female  most  often  deposits  her  eggs.  Caves 
are  also  selected.  Blood  seems  necessary  for  the  fertilization  of  the 
eggs  but  lizard  blood  seems  more  common  in  the  stomach  of  P.  minutus 
than  human  blood.  They  have  also  been  observed  to  feed  on  other 
reptilian  bloods.  The  female  insect  has  been  kept  alive  in  captivity 
up  to  forty-six  days. 

Culicidae. — Mosquitoes  have  three  main  parts  of  the  body — the  head, 
the  thorax,  and  the  abdomen.  On  the  head,  the  space  behind  the  two 
compound  eyes  is  called  the  frons,  in  front,  and  the  occiput  posteriorly. 

The  nape  is  back  of  the  occiput.  The  bulbous  prolongation  of  the  frons  which 
projects  over  the  attachment  of  the  proboscis  is  the  clypeus.  The  clypeus  is  hairy 
in  the  Culex;  scaly  in  Stegomyia.  The  proboscis  is  straight  in  all  mosquitoes  of 
importance  medically.  It  consists  of  a  fleshy,  scaled,  gutter-shaped  portion  be- 
neath, known  as  the  labium,  which  terminates  in  two  hinge-joint  processes — the 
labella.  At  the  end  of  the  labium  is  a  thin  membrane  (Button's  membrane).  It 
is  through  this  that  filarial  embryos  are  supposed  to  pass  on  their  way  from  the 
interior  of  the  labium  to  enter  the  person  bitten.  The  labium  may  be  considered 
as  the  sheath  of  a  knife,  holding  and  protecting  the  slender,  blade-like  penetrating 
organs.  Lying  in  this  groove  we  have,  from  above  downward,  the  horseshoe- 
shaped  labium -epipharynx,  the  undersurface  of  which  is  open.  This  when  closed 
by  the  underlying  hypopharynx  forms  a  tube  through  which  the  blood  is  sucked 
up  by  the  mosquito.  In  the  hypopharynx,  which  somewhat  resembles  a  hypoder- 
mic needle,  is  a  channel,  the  veneno-salivary  duct.  It  is  down  this  channel  that 
the  malarial  sporozoite  passes.  There  are  two  pairs  of  mandibles  and  two  pairs 
of  maxillae  on  either  side  of  the  hypopharynx — the  mandibles  above  and  the  maxillae 
below.  The  serrations  of  the  maxillae  are  coarser  than  those  of  the  mandibles. 
The  sensory  organs,  the  palps,  lie  on  either  side  of  and  slightly  above  the  proboscis. 
These  are  of  the  utmost  importance  in  differentiating  mosquitoes  and  must  not 
be  confused  with  the  antennae,  which  are  attached  above  the  palpi  and  at  the  sides 
of  the  clypeus.  These  antennae  are  of  importance  in  distinguishing  the  sex  of  the 
mosquito. 

The  thorax  is  largely  made  up  of  the  mesothorax,  at  the  posterior  margin  of 


366 


THE   MOSQUITOES 


•r  trilo- 


which  is  a  small,  sharply  defined  piece,  the  scutellum;  this  may  be  smooth  or 
bed.  Underneath  and  posterior  to  the  scutellum  is  the  metanotum;  the  metano- 
tum  is  bare  in  Anophelinae  and  Culicinae,  has  hairs  in  Dendromyinae  and  scales  in 
Joblotinae. 

There  is  a  pair  of  wings  attached  to  the  posterior  part  of  the  mesothorax  and, 
more  posteriorly  still,  a  pair  of  rudimentary  wings  (halteres)  attached  to  the 
metanotum. 

The  wing  venation  is  important.  The  costa  shows  as  a  stout  rib 
or  vein  bordering  the  upper  side  of  the  wing  and  running  around  the. 
apex  and  lower  border. 


FIG.  91. — Anatomy  of  the  mosquito.     No.  7  shows  various  types  of  scales. 

Below,  it  has  a  fringe  which  may  show  spots.  The  location  of  the  spots  in  the  upper 
part  of  the  costa  of  anophelines  is  of  great  value  in  differentiating  species.  Beneath 
the  upper  costal  border  the  subcostal  vein  runs  to  join  the  costa  at  some  distance 
within  the  apex.  The  apex  is  the  free  end  of  the  wing  and  the  base  that  attached 
to  the  thorax.  Running  parallel  to  the  subcosta,  but  reaching  the  apex,  is  the  ist 
longitudinal  vein.  Below  that  is  the  2d  longitudinal  vein  which  forks  to  make 
the  ist  fork  cell  or  ist  submarginal  cell.  The  3d  longitudinal  takes  origin  at  the  junc- 
tion of  the  supernumerary  and  midcross  veins.  The  4th  longitudinal  divides  to 
form  the  2d  fork  cell  (zd  posterior  cell).  The  5th  and  6th  longitudinal  veins 
arise  from  the  base  of  the  wing  and  run  to  the  periphery. 


ANATOMY  OF  THE  MOSQUITO 


367 


The  three  pairs  of  legs  are  attached  to  the  thorax. 

Each  leg  has  9  parts.  The  two  short  ones  are  the  basally  placed  coxa  and  the 
small  trochanter  attached  to  it.  Then  come  the  long  femora,  tibia  and  metatarsi 
with  the  four  segments  of  the  tarsi  terminally.  The  last  tarsal  segment  ends  in  two 
claws,  which  in  the  female  may  be  simple  or  uni-serrated. 

There  are  nine  segments  in  the  abdomen.  The  genitalia  arise  from  the  terminal 
segments  as  bilobed  processes.  In  the  male  there  is  a  pair  of  hook-like  appendages 
or  claspers,  between  which,  and  ventrally  situated,  are  the  harpes,  also  a  pair  of 
chitinous  processes. 


FIG.  92. — Distinguishing  characteristics  of  mosquito  larvae  and  fly  antennas 
Siphon  tubes  of  i,  Stegomyia,  2,  Culex,  3,  Tcsniorhynchus;  mental  plates  of  4,  Tcenio- 
rhynchus,  5,  Stegomyia,  6,  Culux;  larval  antennae  of  7,  Culex,  8,  Stegomyia,  9,  Anoph- 
eles; antennae  of  10,  Muscidae,  n,  Tabanidae,  12,  Simulidae,  13,  Sarcophagidse. 

In  considering  the  question  of  the  possible  danger  which  might  arise  from  the 
introduction  of  a  case  of  yellow  fever,  malaria,  or  filariasis,  it  would  give  the  greatest 
information  if  mosquito  ova  were  at  hand  so  that  we  could  by  watching  the  develop- 
ment from  egg  to  larva,  pupa,  and  insect,  have  all  the  points  from  which  to  decide  as 
to  the  genera  developing  in  the  given  locality.  It  is  generally  a  very  easy  matter 
to  dip  out  large  numbers  of  larvae  from  the  pools  and  having  noted  the  characteristics 
of  the  larvae,  to  do  the  same  when  the  pupae  develop;  so  that  we  have  only  to  verify 
our  identification  when  the  insect  emerges  from  the  pupa. 

THE  OVA 

The  egg  raft  of  Culex,  containing  about  250  ova,  is  quite  perceptible  on  the  surface 
of  the  water  as  a  black,  scooped-out  mass,  about  ^  inch  in  length.  The  eggs 


368 


THE    MOSQUITOES 


are  set  vertically  in  the  raft.     The  eggs  of  the  Stegomyia  are  laid  singly  and  have 
a  pearl-necklace-like  fringe  around  them. 

The  Anophelinae  eggs  are  oval  in  shape  with  air-cell  projections  from  either  side. 
They  are  laid  in  triangle  and  ribbon  patterns.  The  markings  of  these  air  cells  vary 
and  have  been  used  for  differentiation.  The  length  of  time  of  the  egg  stage  varies 
according  to  temperature  and  other  conditions — one  to  three  days  for  Stegomyia 
and  two  to  four  days  for  Anophelina.  The  Anophelinae  are  more  difficult  to  raise 
than  Culex  or  Stegomyia. 

LARV.E 


There  are  two  great  classes  of  larvae — the  siphonate  and  the  asiphonate. 
latter  are  always  Anophelinae. 


The 


STIGMATlC 


"   UATERAL.    ABDOM.  HXVIRS 


ANTENNA 


MOUTH  BRU 


FIG.  93. — Asiphonate  larva.     Anopheles.     2.  Siphonate  larva.     Stegomyia. 


The  Culicinae  larvae  have  a  projecting  breathing  tube  at  the  posterior  extremity 
which  is  called  a  respiratory  siphon.  This  projects  off  at  an  angle  from  the  axis  of 
the  body,  the  true  end  of  which  terminates  in  four  flap-like  paddles.  If  you  divide 
the  length  of  the  siphon  by  the  breath,  you  get  what  is  known  as  the  siphon  index. 
In  Culex  the  siphon  is  long  and  slender,  in  Stegomyia  it  is  short  and  barrel-shaped. 
When  at  the  surface  the  Culex  larva  has  its  siphon  almost  vertical  and  the  body  at  an 
angle  of  about  45°. 


MOSQUITO  LARV2E 

The  Stegomyia  larva  hangs  more  vertically.  As  a  rule,  the  hairs  proceeding 
from  the  sides  of  Culex  larvae  are  straight  and  the  head  relatively  large.  There  are 
also  no  palmate  hairs  along  the  sides. 

The  Anophelinae  larvae  have  a  small  head  which  is  capable  of  being  twisted  around 
with  lightning-like  rapidity.  They  are  darker  in  color  and  have  no  siphon;  float 
parallel  to  the  surface  of  the  water;  have  long  lateral  branching  hairs,  and  on  the 
sides  of  each  of  the  five  or  six  middle  abdominal  segments  they  have  a  pair  of 
palmate  hairs.  These  palmate  hairs  are  supposed  to  aid  them  in  keeping  their  posi- 
tion on  the  surface  of  the  water.  The  larva  are  usually 
called  "wrigglers."  The  duration  of  the  larval  stage  is 
from  one  to  two  weeks,  according  to  the  temperature. 

It  has  been  proposed  to  use  larval  character- 
istics in  differentiating  species  but  as  the  larva 
moults  about  three  times  and  as  the  hairs  or 
spines  of  the  exo-skeleton  of  these  different  larval 
stages  vary  in  number  and  appearance  such  a 
scheme  has  not  met  with  general  approval. 

THE  PUP.E 

The  pupa  of  the  mosquito  is  an  obtected  one 
there  being  only  a  closely  applied  chitinous  coating 
covering  it;  it  does  not  have  a  puparium  as  does 
the  coarctate  pupa  of  the  house  fly.  The  mos- 
quito pupa  is  lighter  than  water  while  the  larva 
is  heavier. 

Pupae  have  a  bloated-looking  cephalo-thorax  and  a 
shrimp-like  tail — the  latter  the  abdomen.  Very  impor- 
tant in  examining  them  with  a  lens  is  to  note  the  char- 
acteristics of  the  siphon  tubes  which  project  from  the 
dorsal  surface.  These  siphons  are  long  and  slender  in 
Culex  and  project  from  the  posterior  portion  of  the  head 
end.  In  Anophelinae  they  are  broadly  funnel-shaped  and  arise  from  the  middle 
of  the  head  end.  The  siphon  of  the  Stegomyia  is  triangular. 

The  bulbous  end  of  the  Culex  nymph  is  more  vertical  than  the  horizontally  placed 
cephalo-thorax  of  Anopheles.  The  duration  of  pupal  life  is  short — only  one  to  three 
days.  At  the  end  of  this  time  the  pupa  comes  to  the  surface  and  straightens  out. 
The  integument  then  splits  dorsally  and  the  perfect  insect  emerges.  It  dries  its 
wings  for  a  time  on  its  raft-like  pupal  skin  and  then  flies  away. 

From  the  above  it  will  be  seen  that  the  stages  in  the  metamorphosis  of  the  mos- 
quito take  about  two  weeks:  one  to  three  days  for  egg  stage;  seven  to  ten  days  for 
larval  stage,  and  two  to  three  days  for  pupal  stage. 


FIG.  94. — Pupae:  i. 
Culex;  2.  Anopheles; 
3.  Aedes  calopus. 
(After  Howard.}  From 
P.  H.  Reports. 


24 


370 


THE   MOSQUITOES 


DISSECTION  OF  THE  MOSQUITO 


The  easiest  way  to  secure  a  mosquito  for  dissection  is  to  use  an  ordinary  plugged 
test-tube.  Slipping  the  open  end  of  the  test-tube  over  the  resting  mosquito,  by  a 
slight  movement,  the  insect  will  fly  toward  the  bottom.  Then  quickly  insert  the 
plug.  If  it  is  not  desired  to  study  the  scales,  the  best  way  to  kill  the  mosquito  is  by 
striking  the  tube  sharply  against  the  thigh.  If  it  is  also  desired  to  study  the  scale 
characteristics  it  is  better  to  put  a  drop  or  so  of  chloroform  on  the  lower  part  of  the 
cotton  plug.  The  vapor  falls  to  the  bottom  of  the  tube  and  kills  the  mosquito.  Take 
the  mosquito  out,  pull  off  legs  and  wings,  and  then  place  the  body  in  a  drop  of  salt 


FIG.  95. — Heads  of  mosquitoes:  i  and  2,  male  and  female  Cttiex  pungens',  3  and  4, 
male  and  female  Anopheles',  5  and  6,  male  and  female  A'edes  calopus.  (After  Stitt.) 
From  P.  H.  Reports. 

solution  on  a  slide.  It  has  been  recommended  to  smear  the  surface  of  the  slide  with 
bile,  wiping  off  the  excess,  before  commencing  the  dissection  in  the  salt  solution. 
Then  hold  the  anterior  end  of  the  thorax  by  pressure  of  a  needle.  With  a  second 
needle  in  the  other  hand,  gently  crush  the  chitinous  connection  between  the  sixth  and 
seventh  segments  of  the  abdomen.  Then  holding  the  thorax  firm,  steadily  and 
gently  pull  the  last  segments  in  the  opposite  direction.  If  this  is  done  properly,  a 
delicate  gelatinous  white  mass  will  slowly  float  out  in  the  salt  solution.  One  should 


CULICINE  AND  ANOPHELINE  MOSQUITOES  371 

be  able  to  secure  the  alimentary  canal  as  far  up  as  the  proventriculus,  which  is  just 
anterior  to  the  stomach,  the  part  in  which  the  malarial  zygotes  develop.  Proceeding 
from  before  backward,  we  have  the  proventriculus,  which  is  a  sort  of  muscular  ring 
at  the  opening  of  the  stomach  or  mid-gut  and  marks  the  separation  of  the  stomach 
from  the  cesophagus.  Opening  into  the  lower  part  of  the  oesophagus  are  the 
cesophageal  diverticula  or  crops,  which  are  food  reservoirs.  Occasionally  in  a 
dissection  we  pull  out  these  structures  which  are  three  in  number. 

Leading  from  the  stomach  we  have  the  hind-gut,  which  ends  in  the  rectum. 

This  has  a  posterior  dilatation  or  rectal  pouch  which  usually  has  three  or  four 
rather  marked  anal  papillae. 

Taking  origin  at  the  posterior  end  of  the  stomach  and  festooning  the  hind-gut 
are  five  longitudinal  tubes — the  Malpighian  tubules.  These  are  characterized  by 
large  granular-like  cells  with  a  prominent  refractile  nucleus.  They  are  regarded  as 
the  renal  structures.  It  is  in  these  tubules  that  the  embryo  of  the  Filaria  immitis 
of  the  dog  develops.  In  the  female  mosquito,  the  parts  withdrawn  may  seem  to  be 
largely  made  up  of  the  white  oval  ovaries.  These  are  connected  with  the  sperma- 
thecae,  in  which  the  spermatozoa  are  stored  after  fecundation  by  the  male.  In  the 
male  the  testicles  are  quite  distinct.  Next  to  the  examination  of  the  stomach  for 
zygotes,  which  appear  as  wart-like  excrescences  on  its  outer  surface,  the  most 
important  structures  are  the  salivary  glands,  where  the  malarial  sporozoites  are  found. 
The  easiest  way  to  dissect  out  the  salivary  glands  is  to  press  down  firmly,  but  gently, 
on  the  anterior  part  of  the  thorax,  and  then  with  the  shaft  of  a  second  needle, 
pressing  on  the  head  to  gently  draw  the  head  away  from  the  thorax,  so  that  by  this 
expression  and  traction  movement  you  extract  them  with  the  head  segment.  They 
are  very  minute  and  are  to  be  told  by  their  exceedingly  highly  refractile  appearance. 
To  examine  for  sporozoites  cover  the  glands  protruding  from  the  neck  with  a  cover- 
glass  and  search  with  a  one-sixth  objective  for  narrow,  curved  bodies  in  the  sub- 
stance of  the  glands.  If  present  try  to  smear  out  the  glands  between  the  cover-glass 
and  slide  by  pushing  the  cover-glass  along,  then  withdrawing  the  cover-glass,  dry 
quickly  and  stain  the  smear  on  slide  or  cover-glass  with  Wright's  stain. 

The  sporozoites  are  narrow  falciform  bodies  about  12/1  in  length,  with  a  central 
chromatin  dot. 

A  matter  about  which  there  is  dispute  is  as  to  whether  the  salivary  glands  com- 
municate with  the  alimentary  canal.  Theobald  states  that  there  is  no  connection 
between  them. 


DIFFERENTIATION  OF  CULICINE  AND  ANOPHELIN^E 

It  is  impossible  even  for  an  entomologist  to  differentiate  mosquitoes 
without  recourse  to  elaborate  keys  and  tables.  It  is  a  comparatively 
easy  matter,  however,  to  decide  as  to  whether  the  mosquito  is  a  prob- 
able malaria  transmitter  or  not. 

While  certain  characteristics  of  the  male  are  used  to  separate  the  .Edina;  from  other 
subfamilies,  yet  it  is  only  with  the  female  that  we  concern  ourselves  in  differentiating 
the  Culicinaj  from  the  Anophelinae.  Therefore,  it  is  first  necessary  to  distinguish 


372 


THE    MOSQUITOES 


the  male  from  the  female.  If  the  antennae  have  not  been  torn  off,  this  can  be  decided 
by  the  highly  adorned  plumose  antennae  of  the  male,  those  of  the  female  being 
sparsely  decorated  with  short  hairs.  The  palpi  of  the  Anophelinae  tend  to  be 
clubbed,  while  those  of  the  Culex  are  straight.  If  the  antennae  have  been  broken  off, 
look  for  the  claspers  at  the  end  of  the  abdomen. 

Male  mosquitoes  do  not  feed  on  blood  but  on  fruits  and  flowers  in- 
stead. The  puncturing  parts  of  the  male  are  not  sufficiently  resistant 
to  penetrate  the  skin. 

Having  determined  that  the  insect  is  a  female,  we  then  proceed  to  place  it  either 
in  the  subfamily  Culicinae  or  Anophelinae  by  a  study  of  the  relative  length  of  the 
palpi  to  the  proboscis.  If  the  palpi  are  shorter  than  the  proboscis,  it  belongs  to  the 
Culicinae;  if  as  long  or  longer,  to  the  Anophelinae.  The  palpi  of  the  female 
Megarhininae  are  also  long,  but  the  proboscis  is  curved. 

Having  settled  on  the  subfamily,  we  separate  the  genera  by  con- 
sidering such  points  as  character  and  distribution  of  scales  on  back  of 
head,  wings,  thorax,  and  abdomen ;  banding  of  proboscis,  legs,  abdomen, 
and  thorax,  shape  of  scales  on  wings,  and  location  of  cross  veins. 


FIG.  96. — Resting  posture  of  mosquitoes:  i  and  2,  Anopheles;  3  Culex  pipienes. 
(After  Sambon.)     From  P.  H.  Reports. 

Anophelinae  show  abundant  upright  forked  scales  on  occiput.  The  mesothorax 
shows  sparse  hairs  or  scales  with  a  smooth  scutellum.  As  a  rule,  the  wings  are  spotted 
(dappled)  and  the  location  of  these  spots  give  the  best  clue  to  the  different  species  of 
the  genera.  With  the  exception  of  Bironella  the  first  submarginal  cell  is  large.  This 
cell  is  longer  than  the  second  posterior  one. 

In  the  resting  position  Culex  allows  the  abdomen  to  droop,  so  that  it  is  parallel 
to  the  wall.  The  angle  formed  by  the  abdomen  with  head  and  proboscis  gives  a 
hunchback  appearance. 

Anopheles  when  resting  on  a  wall  goes  out  in  a  straight  line  at  an  angle  of  about 
45°.  It  resembles  a  bradawl. 

Classification 

There  are  four  subfamilies  of  Culicidae,  differentiated  according  to 
the  palpi: 


MALARIAL    TRANSMITTERS 


373 


1.  Palpi  as  long  as  proboscis  in  females;  proboscis 
straight.     Anophelina. 

2.  Palpi  as  long  or  shorter  than  proboscis  in  females; 
proboscis  curved.    Megarrhinina. 

3.  Palpi  shorter  than  proboscis  in  females.  Culicina. 
Palpi  shorter  than  proboscis  in  male  and  female. 


i .  Scales  on  head  only;  hairs 
on  thorax  and  abdomen. 


Palpi  as   long  or    longer 
than  proboscis  in  male. 


The  important  ones  from  a  medical  standpoint  are  the  Anophelinae 
and  Culicinae. 

Anophelinae 

Scales  on  wings,  large  and  lanceolate.    A  nopheles. 
Palpi  only  slightly  scaled. 

Wing  scales  small  and  narrow  and  lanceolate. 
Myzomyia.     Only  a  few  scales  on  palpi. 
Large  inflated  wing  scales.     CydoUppteron. 
2.  Scales  on  head  and  thorax    f 
(narrow    curve    scales). 
Abdomen  with  hairs. 

.? 

1.  Abdominal    scales    only    on    ventral    surface. 
Thoracic      scales      like      hairs.    Myzorhynchus. 
Palpi  rather  heavily  scaled. 

2.  Abdominal    scales    narrow,    curved   or    spindle- 
shaped.     Abdominal  scales  as  tufts  and  dorsal 

patches.    Nyssorhynchus. 

3.  Abdomen  almost  completely  covered  with  scales 
and  also  having  lateral  tufts.    Cellia. 

4.  Abdomen  completely  scaled.     Aldrichia. 


i.  Wing  scales  small  and  lanceolate.    Pyretophorus. 


Scales  on  head  and  thorax 
and  abdomen.  Palpi 
covered  with  thick  scales. 


NOTE.  —  Of  the  above  genera  only  CydoUppteron  and  Aldrichia  are  unproven 
malarial  transmitters. 

The  following  species  of  anophelines  selected  from  the  different 
genera  are  important  transmitters  of  malaria. 

Anopheles  maculipennis.  —  Wings  with  four  spots  located  at  bases  of  both  forked 
cells  and  of  second  and  third  longitudinal  veins.  No  costal  spots.  Palpi  yellowish 
brown  and  unbanded.  Legs  unbanded. 

Myzomyiafunesta.  —  Wings  with  four  yellow  spots  on  a  black  costa  and  two  black 
line  spots  on  third  longitudinal  vein.  Palps  with  three  white  rings.  Proboscis  un- 
banded. Legs  with  faint  apical  bands. 

Pyretophorus  costalis.—  Costa  black  with  five  or  six  small  yellow  spots.  Palps 
with  two  narrow  white  bands  and  white  tip.  Femora  and  tibias  with  yellow  spots. 
Apical  tarsal  bands. 

Myzorhynchus  pseudopictus.  —  Black  costa  with  two  pale  yellow  spots.  Wing 
fringe  unspotted.  Black  palps  with  four  pale  bands.  Apex  of  palps  white. 

Nyssorhynchus  fuliginosus.  —  Black  costa  with  three  large  yellow  spots. 
Numerous  black  dots  on  the  longitudinal  veins.  Palpi  black  with  white  tip  and  two 
narrow  white  bands.  Last  three  hind  tarsal  segments  white. 

Cellia  argyrotarsis.  —  Black  costa  with  two  distinct  and  several  smaller  white  spots. 


374 


THE   MOSQUITOES 


i .  Posterior  cross  vein  nearer 
the  base  of  the  wing  than 
the  mid-cross  vein. 


Dark  brown  palps  with  two  narrow  bands  and  a  white  tip.    Legs  with  last  three 
hind  tarsal  segments  white. 

The  Megarhinin®  are  of  no  importance  medically. 

The  genus  Megarhinus  has  the  following  characteristics: 

1.  Large  mosquitoes  with  brilliant  metallic  coloring.     (Elephant  mosquitoes.) 

2.  Long,  curved  proboscis. 

3.  Caudal  tufts  of  hairs  on  each  side  of  abdomen. 

The  JEdinae  are  not  known  to  play  any  r61e  in  transmission  of  diseases.  This 
subfamily  is  characterized  by  having  the  maxillary  palpi  much  shorter  in  both  males 
and  females  than  the  proboscis. 

One  genus  Sabethes  is  very  characteristic,  owing  to  dense  paddle-like  scale  struc- 
tures on  two  or  more  legs. 

Differentiation  of  Culicinae  Genera 

1.  Proboscis  curved  in  female.    Psorophora. 

2.  Proboscis  straight  in  female. 

A.  Palps  with  three  segments  in  the  female. 

(a)  Third  segment  somewhat  longer  than  the 
first  two.     Culex. 

(b)  The  three  segments  equal  in  length.     Stego- 
myia. 

B.  Palps  with  four  segments  in  the  female. 

(a)  Palps  shorter  than  the  third  of  the  proboscis. 
Spotted  wings.     Theobaldia. 

(b)  Palps  longer  than  the  third  of  the  proboscis. 
Irregular  scales  on  wings.     Mansonia. 

C.  Palps  with  five  segments  in  the  female.   Tcenior- 

hynchus. 

2.  Posterior  cross  vein  in  line  with  mid-cross  vein.    Joblotina. 

3.  Posterior  cross  vein  further  from  base  of  wing  than  mid-cross  vein.    Muddus. 

Of  the  Culicinae  the  genus  Stegomyia  is  of  importance  on  account 
of  yellow  fever.  The  totally  efficient  hosts  for  filariasis  (filarial  embryos 
found  in  the  thorax  and  proboscis)  are  chiefly  among  the  genus  Culex. 
The  genera  Mansonia  and  Taniorhynchus  may  also  transmit  filariasis. 
Some  think  the  Anophelinae  genera  Cellia  and  Myzomyia  may  transmit 
filariasis  as  well  as  malaria. 

The  genus  Culex  is  implicated  in  dengue. 

Stegomyia. — This  is  the  most  important  culicine  genus.  These  are  mosquitoes 
with  silver  markings.  The  head,  entirely  covered  with  flat  scales,  has  also  some 
upright  forked  scales.  Scutellum  has  dense  flat  scales.  S.  calopus  is  deep  blackish- 
brown  with  two  thoracic  parallel  lines  with  curved  silver-white  lines  outside  (lyre 
marking).  Banding  of  thorax,  abdomen,  and  legs. 

S.  calopus  bites  only  at  night  after  the  first  feeding.  The  first  meal  of  blood 
however  may  be  taken  in  the  daytime.  To  become  infected  it  must  take  blood 
from  a  yellow-fever  patient  in  the  first  two  or  three  days  of  the  disease.  After 
sucking  the  blood  of  a  yellow-fever  patient  the  mosquitoes  cannot  transmit  the 


MALE  AND  FEMALE  MOSQUITOES 


375 


•      ^ 


FIG.     97.— Anopheles     maculipennis        FIG.     98.— Aedes  calopus,  male.     From 
(quadrimaculatus} ,    male.     (After    Cas-  P.  H.  Reports. 

tellani  and    Chalmers.}     From    P.    H. 
Reports. 


FIG.     99. — Anopheles      maculipennis        FIG.  100. — Aedes  calopus,  female.     From 
(quadrimaculatus},  female.      (Castellani  P.  H.  Reports. 

and    Chalmers,    after    Austen.}     From 
P.  H.  Reports. 


376 


THE   MOSQUITOES 


disease  by  biting  a  nonimmune  to  yellow  fever  for  a  period  of  twelve  days.  After 
this  time  the  mosquito  remains  infective  for  its  life — in  one  instance  fifty-seven  days. 

S.  sGutdlaris  has  a  single  silver  stripe  down  the  center  of  thorax.  Mosquitoes 
of  this  genus  are  often  called  "Tiger  mosquitoes."  The  larvae  have  short,  barrel- 
shaped  siphons.  They  breed  particularly  in  receptacles  about  the  house. 

S.  pseudoscutellaris,  which  resembles  S.  scutdlaris,  but  has  white  bands  only,  at 
the  sides  of  the  abdominal  segments,  is  thought  to  transmit  filariasis  in  Fiji. 

Culex. — Male  palpi  long  and  acuminate.  Head  has  narrow  curved  and  upright 
forked  scales.  Laterally,  flat  scales.  C.  fatigans  supposed  to  carry  dengue  as  well 
as  Filaria  bancrofti.  It  also  transmits  Proteosoma  of  birds,  the  life  history  of  which 
in  this  mosquito  paved  the  way  to  the  epochal  discoveries  in  connection  with  malarial 


FIG.  101. — Culex  pungens,  male.    (After 
Howard.)     From  P.  H.  Reports. 


FIG.  102. — Culex  pungens,  female.    (After 
Howard.}     From  P.  H.  Reports. 


transmission  by  anophelines.  This  is  a  brown  mosquito  with  pale  yellow  banding 
of  each  abdominal  segment.  The  legs  are  brown  except  for  the  coxae  and  femora. 

Theobaldia. — These  Culicinae  have  spotted  wings  resembling  Anophelinae. 
These  spots  are  due  to  aggregations  of  scales,  not  to  dark  scales.  Male  palps  are 
clubbed  (like  Anopheles). 

Mucidus. — This  genus  has  a  mouldy  look  from  long  twisted  gray  scales.  The 
legs  are  densely  scaled. 

Mansonia. — This  genus  is  characterized  by  broad  flat  asymmetrical  wing  scales. 
As  the  wing  scales  are  brown  and  yellow  the  wings  are  mottled. 

Grabhomia. — Wings  have  pepper-and-salt  appearance  with  short  fork  cells. 

Taniorhynchus. — This  genus  is  characterized  by  dense  wing  scales,  which  are 
broadly  elongated  with  truncated  apex. 

Acartomyia, — Much  like  Grabhamia,  but  scales  of  head  give  ragged  appearance. 
Male  palpi  clubbed. 

A.  zammittii  was  supposed  to  be  concerned  in  Malta  fever,  but  it  is  known  now 
that  transmission  is  by  medium  of  milk  of  infected  goats. 


CHAPTER  XXII 
POISONOUS  SNAKES 

SNAKES  belong  to  the  class  Reptilia  and  the  order  Ophidia.  The 
two  families  to  which  poisonous  snakes  belong  are  the  colubrine  snakes 
(Colubridae)  and  viperine  snakes  (Viperidae). 

Of  the  Colubridse  the  Hydrophinae  or  sea-snakes  with  rudder-like  compressed  tail 
and  the  Elapinae  with  round  tails  are  most  important. 

Many  of  our  harmless  snakes  such  as  the  garter-snake  and  blacksnake  belong  to 
the  Colubridae. 

The  cobras  belong  to  the  subfamily  Elapinae  and  are  best  known  by  a  neck-like 
expansion  or  hood.  The  only  poisonous  colubrine  snakes  in  the  United  States  are 
the  beadsnake  (Elaps  fuhius)  often  called  the  Florida  coral  snake,  and  the  sonoran 
coral  (Elaps  euryxanthus). 

The  beadsnake  is  black  with  about  seventeen  broad  crimson  bands,  which  bands 
are  bordered  with  yellow. 

Although  small,  they  are  very  venomous.  The  upper  jaw  has  anteriorly  grooved 
fangs,  which  appendages  are  not  present  in  the  nonpoisonous  coral  snakes,  these 
latter  having  teeth  in  the  upper  jaw  so  that  the  wound  shows  four  rows  of  punctures 
instead  of  two  rows  and  one  larger  puncture  on  each  side  to  mark  the  entrance  of  the 
fangs. 

In  Asia  there  are  many  important  poisonous  colubrine  snakesj  the  cobra  (Naja 
tripudians),  the  King  cobra  (Naja  bungarus)  and  the  Kraits  (Bungarus  fasciatus). 

All  of  the  Australian  poisonous  snakes  are  colubrines. 

The  Viperidae  which  are  characterized  by  a  triangular  head  and  tubular  poison 
fangs  are  the  most  important  poisonous  snakes  in  America.  The  rattlesnake 
(Crotalus),  the  copperhead  (Ancistrodon  contortrix),  and  the  water  moccasin 
(A .  piscivorus)  are  widely  distributed  in  the  United  States. 

There  are  many  harmless  snakes  which  more  or  less  resemble  these  "Pit  Vipers" 
as  the  rattlers,  moccasins,  and  copperheads  are  called.  This  term  refers  to  a  deep 
hole  or  pit  found  on  the  side  of  the  head  between  the  nostril  and  the  eye.  It  is  a 
blind  sac. 

Some  divide  the  Viperidae  into  the  Crotalinae,  which  possess  the  pit  and  the 
Viperinae  which  do  not  have  this  structure. 

The  poison  fangs  are  grooved  or  perforated  and  connected  with  the  poison  glands 
which  resemble  salivary  glands  and  may  be  almost  an  inch  in  length  in  large  snakes. 
The  tongue  is  slender  and  forked  and  is  a  tactile  organ. 

The  jaws  are  remarkable  for  their  great  extensibility,  not  only  vertically,  but 
laterally,  by  the  .ligamentous  connections  of  the  two  halves  of  the  mandible  or  lower 
jaw. 

377 


378 


POISONOUS    SNAKES 


As  the  fangs  are  directed  backward  it  is  necessary  for  the  snake  when  striking 
to  open  widely  the  jaws  and  bend  back  the  neck.  The  fangs  are  then  brought 
forward  and  erected  by  the  spheno-pterygoid  muscles.  The  snake  bite  is  a  com- 
bination of  bite  and  blow.  The  functional  fangs  of  colubrine  snakes  however  are  not 
mobile. 

In  addition  to  the  possession  of  the  pit,  these  vipers  have  a  more  or  less  trian- 
gular head  and  in  particular  a  single  row  of  large  scales  on  the  undersurface  posterior 
to  the  vent  (anus),  while  the  harmless  snakes  show  an  elongated  oval  head  and  two 
rows  of  large  ventral  scales  posterior  to  the  vent. 


FIG.  103. — i,  Single  row  of  scales  posterior  to  vent  (poisonous  snakes — water 
moccasin);  2,  double  row  scales  of  harmless  snake  (Natrix);  3  and  5,  side  and  dorsal 
view  of  head  of  pit  viper;  4  and  6,  side  and  dorsal  View  of  head  of  harmless  snake 
(Natrix);  7  and  9,  bite  puncture  and  skull  of  Elaps;  8  and  10,  bite  puncture  and  skull 
of  harmless  snake. 


In  examining  the  wound  made  by  a  snake  the  two  punctures  of  the 
fangs  indicate  the  bite  of  a  poisonous  snake.  If  these  fang  puncture 
points  are  far  apart  it  shows  that  a  large  snake,  and  probably  one 
capable  of  injecting  a  greater  amount  of  venom  has  given  the  bite. 

When  a  snake  strikes  the  fangs  move  from  the  horizontal  to  the  erect  position, 
the  mouth  being  widely  open.  When  the  fangs  enter  the  jaws  close  and  pressure 
is  exerted  on  the  poison  glands  so  that  the  venom  pours  out. 

The  amount  of  venom  varies  with  the  size  and  condition  of  the  snake,  an  adult 
cobra  yielding  about  i  c.c. 


SNAKE  VENOM 


379 


The  cobra,  after  having  bitten,  remains  attached  for  a  short  time  while  the 
daboia  strikes  with  the  greatest  rapidity  and  immediately  releases  itself. 

Cobra  and  krait  bites  (colubrine  snakes)  produce  more  or  less 
similar  symptoms  such  as  paralysis  of  articulation  with  nausea  and 
vomiting  and  later  paralysis  of  the  respiratory  apparatus.  There  is 
only  an  insignificant  reaction  at  the  point  of  bite. 

The  venom  is  mainly  neurotoxic,  causing  death  by  paralysis  of  cardiac  and  re- 
spiratory centers.  Cobra  venom  is  also  very  haemolytic.  This  hasmolysin  is  acti- 
vated by  the  normal  complement  of  the  serum  of  the  animal  poisoned,  the  hsemolysin 
as  contained  in  the  venom  not  being  toxic  when  alone.  Lecithin  also  has  the 
property  of  activating  the  haemolytic  amboceptor  of  venom. 

In  rattlesnake  bites  (viperine  snakes)  there  is  marked  pain  at  the 
site  of  the  wound  with  much  swelling  and  haemorrhagic  infiltration. 
The  swelling  and  petechial  mottling  spread  up  the  limb  from  the  point 
of  entrance  of  the  venom.  Cold  sweats,  nausea,  weak  heart,  and  syn- 
cope are  common 

Rattlesnake  venom  is  active  chiefly  on  account  of  its  haemorrhagin  or  rather 
endotheliolysin,  which  destroys  the  endothelial  lining  of  blood-vessels. 

Venoms  may  also  contain  proteolytic  ferments  Which  may  account  for  the  softening 
of  muscles  in  snake-bite  cases.  The  toxic  effect  of  the  venom  takes  place  without  an 
appreciable  incubation  period,  hence  different  from  true  toxins. 

The  most  venomous  snakes  seem  to  be  the  sea-snakes  (Enhydrina) .  This  venom 
is  almost  entirely  neurotoxic. 

The  tiger  snake  of  Australia  is  almost  equally  venomous  and  the  krait  (B.  cceruleus) 
next.  The  rattlesnake  is  about  one-fifth  as  venomous  as  the  krait. 

Certain  venoms  greatly  increase  the  coagulability  of  the  blood  so  that  intravas- 
cular  thromboses  may  occur.  It  is  chiefly  with  the  venoms  of  Daboia  and  Bun- 
garus  that  such  thromboses  are  likely  to  occur  and  this  accounts  for  the  almost 
instantaneous  death  which  at  times  results  from  bites  of  such  snakes. 

The  nonspecific  treatment  of  snake-bite  poisoning  is  i.  by  applying  a  tight  ligature 
above  the  site  of  the  bite.  The  ligature,  which  should  preferably  be  a  rubber  band, 
is  to  be  applied  about  a  single  bone  extremity,  not  about  one  with  two  supporting 
bones.  2.  The  making  of  deep  incisions  about  the  fang  punctures  and  thorough 
irrigation  with  a  strong  solution  of  potassium  permanganate.  Rogers  has  recom- 
mended that  the  punctures  be  enlarged  with  a  lancet  and  the  resulting  wound  packed 
with  crystals  of  permanganate. 

Recently  Bannerman  has  shown  that  a  dog  bitten  by  a  cobra  cannot  be  saved 
by  free  incision  and  the  rubbing  in  of  permanganate  crystals.  It  may  however  be 
saved  by  the  immediate  injection  of  10  c.c.  of  a  5%  solution  of  permanganate,  but 
not  if  two  minutes  has  elapsed.  Bites  from  the  daboia  are  fatal,  however  the  per- 
manganate be  applied. 

He  therefore  does  not  consider  the  permanganate  treatment  of  any  practical 
value.  Rogers  thinks  that  Bannerman's  experiments  with  dogs  do  not  give  a  true 


380  POISONOUS    SNAKES 

idea  of  the  value  of  permanganate  because  he  has  had  success  in  experimenting  with 
cats  and  because  it  has  saved  human  lives.  Chromic  acid  injections  (i%)  have  also 
been  recommended. 

Internally  alcohol  does  not  seem  to  be  of  any  value,  in  fact  many  of  the  deaths 
have  been  attributed  to  excessive  ingestion  of  whiskey.  Strychnine  in  large,  almost 
poisonous  doses,  was  highly  recommended  in  Australia  but  the  statistics  seem  to 
make  the  value  of  this  remedy  doubtful. 

Antivenins. — The  active  agents  of  snake  venoms  may  be  either  of  the  nature  of 
haemorrhagins,  neurotoxins,  or  fibrin  ferments.  In  colubrine  snakes  the  neurotoxin 
vastly  predominates  while  with  the  viperines  it  is  the  hsemorrhagin.  Certain 
Australian  snakes  contain  all  three  bodies  in  about  equal  proportion  while  with 
the  rattlesnakes  of  America  it  is  almost  entirely  the  haemorrhagin  which  causes 
the  poisoning.  The  Elaps  of  Florida  is  a  colubrine  snake  and  its  venom  is  neurotoxic 
in  nature. 

The  cause  of  death  in  colubrine  snake  bites  is  chiefly  from  paralysis  of  the  respira- 
tory centers  while  with  the  Pit  Vipers  it  is  chiefly  from  haemorrhages  in  the  vital 
organs.  Antitoxins  have  been  prepared  against  both  viperine  and  colubrine  venoms 
and  these  are  specific,  thus  a  colubrine  antivenin  will  not  be  of  value  against  a 
viperine  bite.  Antivenins  should  be  administered  either  intravenously  or  intra- 
muscularly. The  amounts  recommended  for  injections  to  neutralize  a  fatal  dose 
of  snake  poison  vary  from  100  to  300  c.c.  of  the  antivenin  serum.  There  is  no 
accurate  standardization. 


PART  IV 

CLINICAL  BACTERIOLOGY  AND  ANIMAL  PARA- 

SITOLOGY  OF  THE  VARIOUS  BODY 

FLUIDS  AND  ORGANS 


CHAPTER  XXIII 

DIAGNOSIS  OF  INFECTIONS  OF  THE  OCULAR  REGION 

IT  is  advisable  before  taking  material  for  cultures  or  smears  to 
cleanse  the  nasal  area  of  the  eye-lids,  and  especially  about  the  caruncles, 
with  sterile  salt  solution.  Then,  by  gently  pressing  on  the  lids,  we  may 
be  able  to  get  pure  cultures  of  the  organism  causing  the  infection. 
Normally,  we  may  find  in  the  region  of  the  caruncles  various  skin 
organisms,  especially  staphylococci,  giving  white  colonies. 

The  xerosis  bacillus  and  white  staphylococci  may  be  considered  normal  findings 
in  the  conjunctival  sac.  Streptococci  and  pneumococci  have  also  been  reported 
from  apparently  normal  conjunctival  secretions. 

A  small  particle  of  sterile  cotton,  wound  on  a  toothpick,  with  the  aid  of  a  sterile 
forceps,  makes  an  excellent  swab  for  obtaining  material  for  smears;  the  same  may 
first  be  drawn  over  an  agar  surface  in  a  Petri  dish  in  a  series  of  parallel  lines  of  in- 
oculation before  making  the  smears  on  slide  or  cover-glass. 

When  there  is  a  considerable  discharge,  a  capillary  pipette,  with  a  rubber  bulb, 
may  be  used  to  draw  up  sufficient  material  for  cultures  and  smears.  Be  sure  to 
round  off  the  end  of  the  pipette  in  the  flame  and  not  to  use  a  very  fine  capillary 
tube. 

In  conjunctival  cultures,  plates  of  glycerine  agar,  blood  agar,  or  agar 
plates  smeared  with  blood  are  to  be  preferred,  as  the  Gonococcus  and 
Koch- Weeks  bacillus  will  only  grow  on  blood  or  hydrocele  agar.  The 
diphtheria  and  xerosis  bacilli  grow  well  on  glycerine  agar. 

In  addition  to  the  white  Staphylococcus,  the  Streptococcus  may  be  present  when 
inflammation  of  the  nasal  duct  exists. 

The  Streptococcus  is  at  times  responsible  for  a  pseudo-membranous  conjunctivi- 
tis. The  Staphylococcus  is  as  a  rule  the  cause  of  phlyctenular  conjunctivitis. 


382  EYE  INFECTIONS 

The  Pneumococcus  is  a  fairly  common  cause  of  serpiginous  corneal  ulcerati< 
Active  treatment  is  necessary. 

It  is  now  recognized  as  advisable  to  make  an  examination  for  the 
Pneumococcus  before  performing  operations  on  the  eye  as  serious 
results  may  follow  if  the  Pneumococcus  be  present.  It  is  the  organism 
frequently  found  in  dacryocystitis  and,  in  the  case  of  traumatism,  may 
bring  about  panophthalmitis. 


: 


Corneal  ulcerations  are  not  apt  to  appear  even  with  a  pneumococcal  conju 
tivitis  unless  there  be  an  injury  of  the  epithelium. 

The  B.  xerosis  is  possibly  a  harmless  organism  and  must  not  be  accepted  as 
plaining  an  infection  unless  other  factors  have  been  eliminated.  The  true  diph- 
theria bacillus,  which  the  xerosis  so  much  resembles,  may  cause  a  pseudomembran- 
ous  inflammation. 

The  B.  pyocyaneus  may  cause  severe  purulent  keratitis  as  well  as  conjunctivitis. 
The  pyocyaneus  toxin  appears  to  be  a  factor. 

The  Gonococcus  and  the  Koch- Weeks  bacillus  are  usually  responsible 
for  the  very  acute  cases  of  conjunctivitis.  Both  these  organisms  are 
characteristically  intracellular  and  are  Gram  negative. 

Conjunctivitis  in  the  course  of  epidemic  cerebrospinal  meningitis  has  been 
found  to  be  due  to  the  Meningococcus. 

The  diplobacillus  of  Morax  and  Axenfeld  is  more  common  in  chronic,  rather 
dry  affections  of  the  conjunctiva,  chiefly  involving  the  internal  angle  and  showing 
a  morning  accumulation  of  the  secretion.  The  bacilli  are  found  in  twos,  more 
rarely  in  short  chains.  They  are  generally  free  but  may  be  found  in  phagocytic 
cells.  They  resemble  Friedlander's  bacillus  morphologically  but  do  not  have 
capsules. 

In  cases  of  ozena  with  involvement  of  the  nasal  ducts  Friedlander's  bacillus 
may  be  found. 

Even  in  cases  without  ozena,  capsulated,  Gram-negative  bacilli  of  the  Fried- 
lander  group  have  been  frequently  reported  in  conjunctival  inflammation  and  in 
dacryocystitis  as  well. 

The  nodules  of  the  eye-brows  give  the  most  convenient  area  to  take 
material  from  in  the  diagnosis  of  leprosy,  either  the  fluid  expressed 
after  scraping  or  a  piece  of  tissue  cut  into  sections.  Conjunctival  ul- 
ceration  in  leprosy  may  show  abundant  bacilli  as  is  also  true  of  corneal 
ulceration. 

Ordinarily  it  is  impossible  to  find  tubercle  bacilli  in  tuberculous  conjunctival 
discharges. 

The  discharge  from  a  tuberculous  dacryocystitis  may  show  them  satisfactorily. 
Animal  inoculation  is  preferable  in  the  diagnosis  of  ocular  T.  B.  The  Pneumo- 
coccus is,  however,  the  most  important  organism  in  dacryocystitis — rarely  the  B. 
coli. 


INFECTIONS  OF  THE  OCCULAR  REGION  383 

In  a  gonorrhoeal  ophthalmia  the  secretion  is  much  more  abundant  and  there  is 
an  absence  of  contaminating  organisms,  the  reverse  of  infection  with  the  confusing 
M .  catarrhalis.  As  a  matter  of  fact,  large  numbers  of  M .  catarrhalis  may  be  present 
in  the  conjunctival  secretion  with  only  slight  irritation  being  observable. 

Wherry  has  reported  two  cases  of  ulcerative  conjunctivitis  with  lymphadenitis 
of  cervical  glands,  fever  and  marked  prostration,  due  to  infection  with  B.  tularense, 
occurring  in  persons  who  had  handled  rabbits  which  had  died  of  this  plague-like 
infection.  The  organism  was  first  noted  by  McCoy  in  squirrels  in  California.  The 
symptoms  and  lesions  in  these  animals  are  those  of  plague.  Guinea-pigs  succumb 
after  the  cutaneous  inoculation  of  material  and  show  lesions  markedly  resembling 
plague.  The  organism,  however,  will  only  grow  on  coagulated  egg  yolk,  thus 
differentiating  it  from  B.  pestis.  McCoy  has  noted  that  the  infection  in  squirrels 
may  be  transmitted  by  fleas  (Ceratophyllus  acutus). 

In  keratomycosis  the  cause  has  been  ascribed  to  Aspergillus  fumigatus. 

Certain  fungi  of  the  genus  Microsporum  have  been  thought  to  be 
the  cause  of  trachoma,  as  have  also  certain  bacillary  forms.  One 
should  be  very  conservative  about  reporting  fungi  in  smears  or  cul- 
tures of  external  surfaces. 

The  larval  stage  of  Tania  solium  (Cysticercus  celluloses)  has  a  predilection  for 
eye  as  well  as  brain.  It  is  usually  situated  beneath  the  retina. 

The  question  as  to  the  nature  of  the  so-called  ophthalmic  flukes  is  taken  up  under 
trematodes.  Echinococcus  cysts  have  been  reported  in  the  orbit. 

The  adult  Filaria  loa  tends  at  times  to  appear  under  the  conjunctiva  or  in  the 
subcutaneous  tissue  of  the  eye-lids. 

Fly  larvae  have  been  reported  from  the  conjunctival  sacs  hi  the  helpless  sick, 
species  of  larval  sarcophagids  having  been  reported  as  invading  the  conjunctival 
region  in  purulent  ophthalmias. 

Demodex  may  cause  an  obstinate  blepharitis. 

Prowazek  has  thought  that  certain  fine  dots  within  the  cytoplasm  of  epithelial 
cells,  which  stain  best  by  Giemsa's  method  and  which  he  considered  protozoal  in 
nature,  were  the  cause  of  trachoma.  See  Koch-Weeks  bacillus  and  trachoma 
bodies. 


CHAPTER  XXIV 

DIAGNOSIS  OF  INFECTIONS   OF  THE  NASAL  AND  A 

CAVITIES 


URAL 


IN  taking  mateiial  from  the  nasal  cavities,  for  bacteriological  ex- 
amination, it  is  well  to  wash  about  the  alae  with  sterile  water  and 
then  have  the  patient  blow  his  nose  on  a  piece  of  sterile  gauze  and  take 
the  material  for  culture  or  smear  from  this.  If  the  material  is  purulent 
and  located  at  some  ulcerating  spot,  it  is  best  to  use  a  speculum,  and 
either  touch  the  spot  with  a  sterile  swab  or  use  a  capillary  bulb  pipette 
with  a  slight  bend  at  the  end. 

Normally,  we  find  only  white  staphylococcus  colonies  and  colonies  of  short-chain 
streptococci.  The  M.  tetragenus,  B.  xerosis,  and  Hofmann's  bacillus  are  also 
occasionally  found. 

In  some  cases  of  ozena  we  may  find  an  organism  of  the  Friedlander  type  in  pure 
culture. 

Biscuit-shaped  diplococci,  both  Gram-negative  and  positive,  are  to  be  found 
either  normally  or  in  cases  of  coryza.  M ,  catarrhalis  has  probably  been  frequently 
reported  as  the  Meningococcus.  Still,  the  Meningococcus  has  been  found  in  the 
nasal  secretions  of  patients  with  cerebrospinal  meningitis.  B.  influenza  and  the 
Pneumococcus  have  also  been  frequently  found  in  cultures  from  the  nasal  secretions. 

Diphtheria  involving  the  nasal  cavity  must  always  be  kept  in  mind, 
and  in  quarantine  investigations  the  examination  of  the  nasal  secre- 
tions culturally  should  be  a  part  of  the  routine. 

The  tubercle  bacillus  may  be  found  in  nasal  ulcerations;  it  is,  however,  only 
present  in  exceedingly  small  numbers.  On  the  other  hand,  one  of  the  best  diag- 
nostic procedures  in  leprosy  is  to  examine  smears  from  nasal  mucous  membrane 
for  the  B.  hpra.  In  such  ulcerations  the  bacilli  are  found  in  the  greatest  profusion. 
Rarely  glanders  may  cause  ulcerations. 

B.  proteus  is  frequently  responsible  for  the  production  of  foul  odors  in  nasal 
discharges  but  does  not  seem  to  produce  inflammatory  conditions  of  the  nasal 
mucosa.  It  simply  decomposes  the  discharges.  Various  fungi  have  been  reported 
from  the  nose,  but  in  such  a  region  the  strictest  conservatism  in  reporting  should  be 
observed. 

Recently  sporozoa  have  been  reported  in  a  case  of  nasal  polyp.  (Rhinos poridium.) 

So  many  degenerative  changes  in  epithelial  cells  resemble  protozoal  forms  that 
such  findings  require  ample  confirmation. 

384 


INFECTIONS   OF   THE   NASAL   AND   AURAL   CAVITIES  385 

The  larval  form  of  Linguatula  rhinaria  is  a  rare  parasite  of  the  nasal  cavities; 
it  is  not  infrequent,  however,  in  the  nostrils  of  dogs. 

Various  fly  larvae  are  far  more  common,  and  the  "screw- worm," 
the  larva  of  the  Chrysomyia  macellaria,  is  common  in  certain  parts  of 
tropical  America,  and  may  by  its  burrowing  effects  cause  fatal  results. 

The  larvae  of  Sarcophaga  have  in  particular  been  found  in  the  nasal  cavities  of 
children.  Myriapods,  while  of  very  little  importance  elsewhere,  have  been  reported 
more  than  30  times  from  the  nasal  fossae. 

In  a  study  of  the  bacteriology  of  otitis  media,  in  277  cases,  Libman 
and  Celler  found  streptococci  present  alone  in  81%,  Streptococcus 
mucosus  in  10%  and  the  Pneumococcus  in  8%;  Staphylococcus,  B.  pyo- 
cyaneus  and  B.  proteus  have  also  been  found.  Mixed  infections  are 
common. 

Streptococci  are  the  organisms  which  most  often  cause  sinus  thrombosis  and  brain 
abscess.  The  influenza  bacillus  has  been  reported  as  a  cause  of  acute  otitis  media. 

Nonvirulent  diphtheroid  bacilli  are  not  infrequently  obtained  in  cultures  from 
ear  discharges. 

Other  organisms  which  have  been  isolated  from  middle-ear  or  mastoid  discharges 
are  B-.  coli,  M.  catarr kalis,  M.  tetragenus  and  Friedlander's  bacillus. 

B.  typhosus  may  be  found  in  middle-ear  discharges  of  persons  who  have  had  an 
attack  of  typhoid  fever. 

The  middle  ear  is  normally  free  of  bacteria,  but  in  affections  of  the 
throat,  as  with  streptococci,  pneumococci,  and  diphtheria  bacilli, 
these  organisms  may  infect  it  by  way  of  the  Eustachian  tube. 

The  moulds  are  of  greater  importance  in  affections  of  the  external  auditory  canal 
than  the  bacteria.  The  cerumen  seems  to  make  a  good  culture  medium  so  that 
various  species  of  Aspergillus,  Mucor,  etc.,  develop  and  close  the  canal.  These 
infections  are  often  introduced  by  the  patient's  finger.  Various  mites  and  fly  larvae 
have  been  reported  from  the  ear. 

The  "screw  worm,"  the  larva  of  Chrysomyia  macellaria,  is  the  most  common 
cause  of  aural  myiasis  in  tropical  America.  The  fly  deposits  its  eggs  about  aural 
and  nasal  cavities  of  those  with  offensive  discharges.  The  larvae  develop  and 
cause  intense  pain  and  giddiness.  Larvae  of  Sarcophaga,  Calliphora  and  Anthomyia 
have  also  been  reported  from  the  external  auditory  meatus.  The  tympanic  mem- 
brane may  be  perforated. 


CHAPTER  XXV 
EXAMINATION    OF  BUCCAL  AND  PHARYNGEAL  MATERIAL 

IN  a  preparation  made  from  material  taken  by  a  sterile  swab  from 
the  region  of  the  normal  buccal  and  pharyngeal  cavities  and  stained  by 
Gram's  method  we  are  struck  by  the  variety  of  organisms  present. 
The  fungi  of  thrush  are  best  examined  for  in  a  preparation  of  membrane 
mounted  in  10%  caustic  potash  solution.  Manilla  may  be  found  in 
sprue  ulcerations  about  tongue  or  buccal  mucosa.  In  examining  for 
buccal  amoebae  mount  the  purulent  material  from  pyorrhoea  in  the 


FIG.  104. — Vincent's  angina.     Spirochata  vincenti.     (Coplin.) 

patient's  saliva.     Smears  from  about  carious  teeth  often  show  the  fusi- 
form bacillus  and  delicate  spirillum  of  Vincent  as  well  as  cocci. 

Gram-positive  and  Gram-negative  staphylococci  are  normally  present,  as  are  also 
streptococci,  pneumococci,  leptothrix  forms,  and  very  probably  yeasts  and  sarcinae 
types  with  many  Gram-negative  bacilli.  If  pseudo-diphtheria  organisms  are 
present,  we  have  these  showing  a  Gram-positive  reaction.  If  this  material  is 
smeared  on  agar  plates  and  cultured  at  37°C.,  we  are  struck  by  the  fact  that  the 
colonies  on  the  plates  may  be  exclusively  staphylococcal  and  streptococcal. 

Of  course  diphtheroids  as  well  as  diphtheria  organisms  grow  well  on  ordinary  agar 
plates.  For  Meningococcus  carriers  a  blood  agar  or  starch  agar  plate  is  advisable. 

It  is  very  difficult,  if  not  impossible,  to  distinguish  a  Pneumococcus  colony  from 

386 


EXAMINATION   OF  BUCCAL   AND   PHARYNGEAL   MATERIAL      387 

a  Streptococcus  one  on  a  plate  culture.  The  presence  or  absence,  however,  of  the 
Pneumococcus  is  distinctly  shown  in  the  Gram-stained  smear,  either  by  its  lance- 
shaped  morphology  or  the  presence  of  a  capsule.  It  has  been  my  experience  that 
smears  from  about  15%  of  normal  individuals  show  capsulated  pneumococci. 

In  diphtheria  examinations  we  rely  chiefly  on  the  cultural  findings  on  Loffler's 
serum.  Where  the  process  is  streptococcal  or  due  to  the  organisms  associated  with 
Vincent's  angina,  the  immediate  examination  of  a  smear  from  the  suspected  spot 
or  area  gives  greater  diagnostic  information.  The  Streptococcus  being  so  abundant 
in  cultures  from  normal  throats,  it  is  difficult  to  determine  its  significance  in  a  cul- 
ture; abundance  of  streptococci  in  a  smear  from  an  ulceration  or  bit  of  membrane, 
however,  is  of  etiological  import.  Streptococcal  sore  throats  are  often  very  toxic 
and  may  be  fatal — often  milk  borne.  Use  blood  agar  plates  to  differentiate  hjemo- 
lyzing  and  "viridans"  types  of  streptococci. 

By  staining  with  Neisser's  method  it  is  possible  to  make  an  imme- 
diate diagnosis  of  diphtheria  from  a  smear  from  a  piece  of  membrane 
in  about  25%  of  cases.  It  is  well,  however,  to  always  culture  such 
material.  The  toluidin  blue  stain  of  Ponder  is  the  best  stain  for 
diphtheria. 

Material  from  the  throat  is  ordinarily  best  obtained  with  a  sterile  copper-wire 
cotton-pledget  swab.  The  platinum  loop  usually  bends  too  easily.  A  sterile  for- 
ceps may  be  more  convenient  for  obtaining  particles  of  membrane.  It  is  believed 
that  ulcerative  conditions  of  the  throat,'  associated  with  the  presence  of  the  large 
fusiform  bacillus  and  delicate  spirillum,  which  make  the  picture  of  Vincent's  angina, 
are  more  common  than  is  usually  so  considered. 

In  Giemsa-stained  smears  from  the  dirty  membrane  covering  the  ulcerated  area 
of  Vincent's  angina  there  are  usually  two  types  of  the  fusiform  bacillus  to  be  seen; 
one  rather  slender,  pale  blue  with  maroon  dots  at  either  end,  the  other  rather  thicker 
and  of  a  uniform  maroon  staining.  The  spirilla  are  from  10  to  18  microns  long  and 
the  fusiform  bacilli  from  5  to  7  microns. 

As  a  rule,  only  cultures  on  Loffler  serum  are  made  and  very  rarely  direct  smears. 
If  a  smear  were  always  made  and  stained  by  Gram's  method  (with  a  contrast  stain 
of  dilute  carbol  fuchsin)  at  the  same  time  the  culture  was  made,  it  is  probable 
that  much  information  of  value  would  be  obtained. 

The  B.  fusiformis  is  an  anaerobe  which  gives  a  fetid  odor  but  cul- 
turally has  no  distinct  characteristics.  The  spirillum  has  not  been  cul- 
tivated. It  has  been  thought  that  the  bacillus  and  spirillum  are 
different  stages  of  the  same  organism. 

At  times  aggregations  of  the  fusiform  bacillus  give  the  appearance  of  branching  so 
characteristic  of  diphtheria  organisms.  Being  Gram-negative,  however,  the  differ- 
entiation is  easily  made— the  B.  diphtheria  being  Gram-positive.  Again  the  attenu- 
ated ends  of  the  fusiform  bacillus  are  diagnostic. 

It  is  usually  stated  that  the  fusiform  bacillus  is  nonmotile.  By  mounting  material 
in  saliva  I  have  noted  a  sluggish,  but  distinct  motility.  The  fusiform  bacillus 


388  VINCENT'S  ANGINA 

and  spirillum  are  often  associated  with  cocci  and  amoebae  in  pus  from  dental  caries 
or  pyorrhoea  and  I  mount  such  material  in  the  patient's  saliva  to  obtain  motility 
in  the  amoebae.  The  fusiform  bacillus  is  not  markedly  Gram-negative. 

The  culturing  of  material  from  the  nasopharyngeal  region  of  contacts  as  well  as 
patient  is  very  important  in  outbreaks  of  cerebrospinal  fever.  Use  a  bent  wire 
applicator  with  sterile  cotton  tip  and  pass  it  to  the  nasopharynx  avoiding  the  uvula. 
Inoculate  tubes  of  serum  or  blood  agar  immediately. 

Direct  smears  are  the  procedure  of  choice  in  streptococcal  and 
pneumococcal  anginas  as  well  as  in  Vincent's  angina. 

Unless  very  familiar  with  the  morphology  of  Treponema  pallidum  and  using 
dark  field  or  Fontana's  staining  procedure,  we  should  be  very  conservative  in  report- 
ing such  an  organism  from  suspected  syphilitic  ulcerations  of  the  throat. 

We  now  know  that  we  have  treponemata  in  the  buccal  cavity  similar  to  T.  pal- 
lidum so  that  even  with  the  dark  field  illumination  I  would  base  a  diagnosis  on 
the  clinical  and  Wassermann  reactions  rather  than  morphologically. 

The  thrush  fungus  (Endomyces  albicans)  may  be  easily  demonstrated  in  a  Gram- 
stained  specimen  as  violet  mycelial  structures. 

Yeasts  due  to  food  particles  are  not  infrequently  observed  in  smears  and  cultures 
from  the  mouth. 

Actinomycosis  may  develop  about  a  carious  tooth  and  the  finding  of 
the  ray  fungus  in  the  granules  from  the  pus  may  give  the  diagnosis. 

Amoebae  and  flagellates  have  been  reported  from  the  mouth.  Also  in  the  re- 
markable disease  "halzoun,"  flukes  have  been  found  to  be  the  cause  of  the  asphyxia. 

In  the  tropics,  round  worms  (A  scar  is]  may  be  vomited  up  and,  lodging  in  the 
pharynx,  may  have  to  be  extracted. 

During  the  campaign  of  Napoleon  in  Egypt  many  cases  of  leech  involvement  of 
the  nasal  and  buccal  cavities  were  noted.  The  parasite  was  the  Limnatis  nilotica 
which  gained  access  to  the  upper  pharynx  through  drinking  water  from  springs  and 
pools.  Many  such  cases  continue  to  be  reported  from  the  Mediterranean  basin. 


CHAPTER  XXVI 
EXAMINATION  OF  SPUTUM 

FREQUENTLY  the  material  submitted  for  examination  as  sputum  is 
simply  buccal  or  pharyngeal  secretion,  or  more  probably  secretion 
from  the  nasopharynx,  which  has  been  secured  by  hawking.  It  should 
always  be  insisted  upon  that  the  sputum  be  raised  by  a  true  pulmonary 
coughing  act,  and  not  expelled  with  the  hacking  cough  so  frequently 
associated  with  an  elongated  uvula.  When  there  is  an  effort  to  deceive, 
some  information  may  be  obtained  from  the  watery,  stringy,  mucoid 
character  of  the  buccopharyngeal  material  and  also  from  the  presence 
of  mosaic-like  groups  of  flat  epithelial  cells  (often  packed  with  bacteria). 
The  pulmonary  secretion  is  either  frothy  mucus  or  mucopurulent 
material,  and  if  the  cells  are  alveolar  they  greatly  resemble  the  plasma 
cells.  At  times  these  cells  may  contain  blood  pigment  granules  (heart- 
disease  cells). 

In  the  microscopic  examination  a  small,  cheesy  particle,  the  size  of  a  pin  head, 
should  be  selected.  This  should  be  flattened  out  in  a  thin  layer  between  the  slide 
and  cover-glass  and  should  be  examined  for  elastic  tissue,  heart-disease  cells,  eggs 
of  animal  parasites,  amoebae,  and  fungi.  Echinococcus  booklets,  Curschman  spirals 
besprinkled  with  Charcot-Leyden  crystals,  and  haematoidin  and  fatty  acid  crystals 
may  also  be  observed. 

Curschman  spirals  indicate  bronchial  as  against  cardiac  or  uremic  asthma.  Char- 
cot-Leyden crystals  have  no  special  significance,  except  in  certain  tropical  diseases 
when  these  crystals  often  are  present  in  paragonomiasis  sputum  and  in  the  pus 
of  amcebic  liver  abscesses  discharging  by  way  of  the  lungs. 

It  may  facilitate  the  examination  of  the  sputum  for  elastic  tissue 
and  actinomycosis  and  other  fungi  to  add  10%  sodium  hydrate  to  the 
preparation. 

To  make  smears  for  staining,  the  sputum  should  be  poured  on  a  flat  surface, 
preferably  a  Petri  dish,  and  a  bit  of  mucopurulent  material  selected  with  forceps.  A 
dark  back-ground  facilitates  picking  out  the  particle.  A  toothpick  is  well  adapted 
to  smearing  out  such  material  on  a  slide.  After  using  the  toothpick  it  can  be  burned. 
When  dry,  the  smear  is  best  fixed  by  pouring  a  few  drops  of  alcohol  on  the  slide, 
allowing  this  to  run  over  the  surface,  and  then,  after  dashing  off  the  excess  of  alcohol, 
to  ignite  that  remaining  on  the  film  in  the  flame  and  allow  to  burn  out. 

389 


390 


ANTIFORMIN 


merit 


A  mark  with  a  grease  pencil,  about  ^  inch  from  the  end,  gives  a  convenient 
surface  to  hold  with  the  forceps  and  also  prevents  the  stain  subsequently  used  from 
running  over  the  entire  surface.  A  piece  of  glass  tubing  about  1 2  inches  long  bent 
into  a  narrow  V  shape  makes  a  very  satisfactory  rest  for  the  slide  in  staining  and  is 
convenient  for  the  steaming  of  staining  solution  over  the  flame. 

Sputum  should  as  a  routine  measure  be  stained  by  the  Ziehl-Neelson  method  and 
by  Gram's  method. 

In  examining  for  tubercle  bacilli  it  may  be  necessary  to  employ  some  method  for 
concentrating  the  bacterial  content  of  the  sputum  prior  to  making  the  smear.  A 
very  satisfactory  method  is  that  of  Miihlhauser-Czaplewski.  Shake  up  the  sputum 
with  four  to  eight  times  its  volume  of  %%  solution  of  sodium  hydrate  in  a  stoppered 
bottle.  When  the  mixture  has  become  a  smooth,  mucilaginous-looking  fluid,  add  a 
few  drops  of  phenolphthalein  solution  and  bring  the  pink  mixture  to  a  boil. 

Then  add  drop  by  drop  a  2%  solution  of  acetic  acid,  stirring  constantly,  until 
the  pink  color  is  just  discharged.  If  the  least  excess  of  acid  is  added  over  that  just 
sufficient  to  cause  the  pink  color  to  disappear,  mucin  will  be  precipitated.  Now  pour 
this  mixture  into  a  centrifuge  tube  and  smear  the  sediment  on  a  slide  and  stain  for 
tubercle  bacilli. 

Antifonnin. — Tubercle  bacilli  usually  occur  nested  in  clumps  of 
sputum.  Therefore,  when  few  in  number  it  is  only  by  chance  that  they 
may  be  found.  Concentration  methods  aim  to  dissolve  these  clumps 
of  sputum  and  collect,  free  from  mucus,  whatever  bacilli  may  be 
present.  There  are  many  concentration  methods  for  sputum.  One 
of  these  has  been  given  above.  Uhlenhuth's  method  has  some 
advantages  over  others  in  the  solvent  used:  i.  It  breaks  up  the 
sputum  very  rapidly;  2.  it  immediately  dissolves  all  organisms  except 
acid-fast  ones;  3.  applied  in  not  too  concentrated  form  and  for  not 
too  long  a  time,  tubercle  bacilli  are  not  killed,  so  that  by  washing 
the  sediment  carefully  by  several  dilutions  and  centrifugings  we  have 
in  the  sediment  viable  tubercle  bacilli  which  we  may  attempt  to 
cultivate  upon  Dorsett's  or  other  suitable  media  with  the  reasonable 
hope  that  contaminations  will  not  choke  them  out  or  prematurely 
kill  the  inoculated  guinea-pig;  4.  it  has  less  effect  upon  the  staining 
properties  of  tubercle  bacilli  than  any  other  material  used  in 
concentration  methods.  PetrofTs  method  is  probably  better. 

To  make  this  solvent  (antiformin)  take  double  the  quantity  of  chlorinated  lime 
and  sodium  carbonate  required  by  the  U.  S.  Pharmacopoeia  and  prepare  according 
to  U.  S.  P.  directions.  To  the  finished  liquor  sodae  chlorinatae  (Labarraque's  solu- 
tion) add  7^%  of  sodium  hydrate. 

The  Liquor  sodae  chlorinatae  'of  the  Br.  P.  is  slightly  stronger  and  some  English 
authorities  recommend  a  mixture  of  equal  parts  of  this  Labarraque's  solution  and 
15%  sodium  hydrate  solution.  As  a  rule  i  part  of  antiformin  to  5  parts  of  sputum 
is  sufficient.  Very  tenacious  sputum  may  require  i  part  to  4  parts  of  sputum. 


EXAMINATION  OF  SPUTUM  391 

If  more  antiformin  is  used  the  specific  gravity  is  too  much  increased  and  the  bacilli 
are  damaged.  The  fluidification  is  hastened  at  incubator  temperature. 

To  5  parts  of  sputum  add  i  part  of  antiformin,  shake  well  and  place  in  incu- 
bator for  one  hour.  To  10  c.c.  of  the  homogeneous  mixture  add  1.5  c.c.  of  a 
solution  made  up  of  i  part  chloroform  and  9  parts  alcohol.  Shake  violently  and 
centrifuge  for  fifteen  minutes.  Mix  the  sediment  with  egg  albumin,  smear  out  and 
stain. 

When  it  is  desired  to  culture  the  tubercle  bacilli  mix  20  c.c.  of  sputum  with  65  c.c. 
sterile  water  and  add  15  c.c.  antiformin.  Stir  the  mixture  with  a  glass  rod.  After 
thirty  minutes  to  two  hours  we  should  have  a  homogeneous  mixture.  Centrifuge 
for  fifteen  minutes  or  longer,  wash  the  sediment  twice  with  sterile  salt  solution  and 
smear  out  the  well- washed  sediment  over  serum  or  glycerine  egg  slants.  The  tubes 
should  be  covered  with  black  paper  and  the  plugs  paraffined.  It  must  be  remem- 
bered that  for  culturing  tubercle  bacilli  we  must  protect  the  growth  from  sunlight 
as  this  will  kill  the  organism.  If  fluid  culture  media  are  inoculated  the  transferred 
material  should  be  deposited  on  the  surface.  Should  the  particle  sink  growth  will 
not  occur. 

Sputum  smears  stained  by  some  Romanowsky  method  or  by  the 
haematoxylin-eosin  stain  are  best  adapted  for  the  study  of  various 
cells,  and  in  particular  of  the  eosinophile  cells  so  characteristic  of 
bronchial  asthma.  In  sputum  from  cancer  of  the  lungs  the  large 
vacuolated  cells  may  be  found. 

When  examining  the  sputum  of  the  bronchopneumonia  of  influenza  the  formol 
fuchsin  gives  the  best  results.  The  influenza  bacilli  are  found  in  little  masses,  fre- 
quently grouped  about  small  collections  of  M.  tetragenus.  The  cocci  stain  a  rich 
purplish-red,  while  the  small  influenza  bacilli  take  on  a  light  pink  color. 

A  greenish  yellow,  nummular  sputum,  often  profuse,  is  frequently  noted  in  in- 
fluenza. 

T.  B.  sputum  showing  a  mixed  infection  with  streptococci  or  pneumococci  or  with 
the  influenza  bacillus  makes  for  a  bad  prognosis.  M.  tetragenus,  which  often  is 
present  when  cavities  exist,  does  not  seem  to  be  so  unfavorable  prognostically. 

Red  cells  show  up  well  in  specimens  stained  by  the  Romanowsky  method;  if 
rouleaux  formation  is  marked,  it  may  indicate  pulmonary  infarction. 

In  culturing  sputum  a  mucopurulent  mass  should  be  washed  in 
sterile  water  and  should  then  be  dropped  into  a  tube  of  sterile  bouillon. 
With  a  sterile  swab  it  should  be  emulsified  and  successive  streaks  made 
along  the  surface  of  an  agar,  blood  agar  or  glycerine  agar  plate.  In 
obtaining  cultures  from  influenza  sputum,  first  smear  the  material 
thoroughly  over  a  blood-serum  slant;  then  inoculate,  by  thorough 
smearing  over  the  surface  of  successive  blood-streaked  agar  slants,  the 
material  on  the  surface  of  the  blood-serum  slant.  The  platinum  loop 
should  be  transferred  from  one  slant  to  another  without  recharging. 
The  influenza  bacillus  seems  to  grow  better  if  the  blood-streaked  agar 


392  ALBUMEN  IN  SPUTUM 


11  that 


slants  are  prepared  just  before  inoculating  with  the  sputum.  All 
is  necessary  is  to  sterilize  an  ear,  puncture  and  take  up  the  exuding  blood 
with  a  large  loop  and  smear  over  the  agar  slant.  Cultures  for  tubercle 
bacilli  are  impracticable  except  with  antiformin  or  by  PetrofTs  method. 
This  latter  is  most  satisfactory.  A  guinea-pig  should  be  inoculated. 

The  blood-stained  watery  sputum  of  plague  pneumonia  should  be  cultured  on 
plates  of  plain  agar  and  3%  salt  agar  at  the  same  time.  An  ordinary  smear  stained 
with  carbol  thionin,  however,  practically  makes  a  diagnosis.  Be  sure  to  inoculate 
a  guinea-pig  cutaneously. 

Pneumococci,  M.  catarrhalis,  and  Friedlander's  bacillus  in  sputum  are  best 
demonstrated  by  Gram's  method  of  staining. 

The  distinct  capsule  staining  of  the  pneumococci  in  a  Gram  preparation  of 
sputum  from  a  suspected  case  of  pneumonia  is  of  value  in  diagnosis. 

The  finding  of  the  ray  fungus  (D.  bovis)  in  sputum  gives  the  diagnosis  of  actino- 
mycosis.  Streptothrix  infections  of  lungs  have  been  confused  with  tuberculosis. 

Moulds,  especially  aspergilli,  may  be  found  in  sputum.  Species  of  Mucort 
Cryptococcus,  and  Endomyces  have  also  been  reported. 

Amoebae  from  liver  abscess  rupturing  into  the  lung  may  be  found. 
Very  important  pulmonary  infections  are  those  with  Paragonimus 
westermanii.  This  is  recognized  by  the  presence  of  operculated  eggs 
in  the  sputum. 

A  fluke,  F.  giganlea,  was  once  found  in  sputum. 

Hydatid  cysts,  either  of  the  lung  or  of  the  liver,  rupturing  into  the  lung,  may  be 
recognized  by  the  presence  of  echinococcus  booklets.  The  material  is  bile-stained 
if  from  the  liver.  Dutcher  has  reported  filarial  embryos  from  sputum. 

Strongylus  apri  has  been  reported  once  from  the  lungs  and  embryos  might  be 
found  in  the  sputum.  In  pulmonary  bilharziosis  Schistosoma  egggs  may  be  found  in 
the  sputum. 

The  test  for  ALBUMEN  IN  THE  SPUTUM  is  of  value  in  the  diagnosis  of  pulmonary 
tuberculosis. 

About  10  c.c.  of  fresh  sputum  as  pure  as  possible  from  saliva  is  mixed  with  an 
equal  quantity  of  water  and  2  c.c.  of  a  3%  solution  of  acetic  acid  to  remove  mucin. 
After  filtering  the  filtrate  is  tested  for  albumin.  The  test  is  obtained  also  in 
pneumonia  and  pleurisy  with  effusion. 


CHAPTER  XXVII 
THE  URINE 

MATERIAL -for  staining  is  best  obtained  by  centrifuging  the  urine, 
then  pouring  off  the  supernatant  urine,  then  draining  the  mouth  of 
the  centrifuge  tube  against  a  piece  of  filter-paper  so  that  we  have  only 
the  pus  sediment  to  finally  remove  with  a  capillary  bulb  pipette  or 
toothpick  stuck  in  a  urine  sediment  pipette  and  make  smears. 

I  always  take  up  the  material  with  the  centrifuge  tube  in  a  slanting  position 
following  the  draining  off  of  the  supernatant  urine  to  avoid  urine  admixture  in 
the  smear  as  such  makes  staining  less  satisfactory.  The  Gram  staining  is  most  satis- 
factory, counterstaining  with  bismarck  brown. 

The  addition  of  a  loopful  of  egg  albumen  or  blood  serum  to  about  twice  that 
amount  of  urinary  sediment  gives  better  results.  (See  under  Staining  Methods.) 

In  pathological  urine  there  is  enough  albumin  to  fix  the  smear. 

The  smear  may  be  stained  after  fixing  by  heat  with  Gram's  stain,  T.  B.  stain, 
or  haematoxylin  and  eosin.  The  latter  is  the  best  for  the  staining  of  epithelial  cells 
and  animal  parasites;  the  Gram  method  for  bacteria. 

It  is  frequently  difficult  to  distinguish  the  spores  of  moulds  from  red  blood-cells 
except  by  measurement  and  staining  reactions.  Spores  of  moulds  rarely  exceed 
five  microns. 

Of  the  greatest  value  is  the  finding  of  phagocytized  bacteria  in  the 
pus  cells  of  the  Gram-stained  smear.  These  indicate  the  causative 
organism  which  show  beautifully  in  the  beginning  of  pyelitis  infections. 
To  examine  for  epithelial  cells  I  make  a  vaseline  streak  across  a  slide 
about  %  inch  from  the  center.  A  drop  of  the  sediment  is  deposited 
on  the  slide  which  may  then  be  examined  unstained  with  the  %  inch 
objective  and  then  a  drop  of  Gram's  iodine  solution  is  added.  One 
edge  of  a  square  cover-glass  is  rubbed  into  the  vaseline  line  and  allowed 
to  drop  on  the  fluid  preparation.  Currents  are  avoided  and  the  cells 
stain  up  beautifully. 

It  is  difficult  to  determine  the  presence  of  blood  in  urine  in  higher  dilution  than 
i  to  300  with  the  spectroscope.  The  ordinary  occult  blood  test  will  show  it  in  much 
higher  dilution. 

To  secure  urine  for  bacteriological  examination  catheterization  is  rarely  necessary 
in  men — in  the  case  of  women  it  is  the  proper  method. 

393 


394 


CULTUEING  URINE 


icn  the 


Authorities  generally  insist  upon  a  catheterized  specimen  in  all  cases  when 
urine  is  to  be  cultured.  As  a  matter  of  fact  when  there  is  a  bacterial  infection  the 
specific  organism  is  in  such  predominant  numbers  that  it  is  easily  distinguished 
from  a  possible  contaminator.  Of  course  should  one  culture  the  urine  in  a  tube  of 
bouillon,  before  plating  out,  a  contamination  might  overgrow  the  causative  organism, 
but  one  should  always  plate  directly  from  urine  which  has  just  been  passed.  The 
smear  stained  by  Gram's  also  checks  up,  particularly  if  certain  bacteria  are  found 
phagocytized  in  pus  cells.  The  man  who  follows  the  clinical  side  as  well  as  the  labora- 
tory one  is  rarely  confused  by  an  occasional  contaminating  organism  on  a  plate 
made  from  urine  or  blood.  Of  course  the  problem  is  more  difficult  with  urine, 
but  when  culturing  of  urine  is  a  routine  procedure  the  worker  soon  knows  the  organ- 
isms likely  to  be  encountered  in  urine  of  women  as  well  as  that  of  men.  As  a  matter 


Oxyhemoglobin 


Methemoglobin 


Reduced 
Hemoglobin 


CO  Hemoglobin 


FIG.  105. — Principal  Blood  Spectra.     (Da  Costa.} 


of  fact  I  rarely  find  colonies  on  plates  made  from  the  urine  of  normal  men,  the  only 
precautions  taken  being  those  noted  below.  I  now  use  blood  agar  plates  as  routine 
plating  media. 

The  glans  penis  and  meatus  should  be  thoroughly  washed  with  soap  and  water, 
after  which  dilute  alcohol  (70%)  should  be  used.  The  greater  part  of  the  urine 
first  passed  should  be  rejected  and  only  the  last  portion  passed  should  be  caught 
in  a  sterile  receptacle.  A  drop  of  this  urine  may  be  either  streaked  over  the 
surface  of  an  agar,  blood  agar,  or  a  lactose  litmus  agar  plate,  or  so  treated  after 
being  first  diluted  in  a  tube  of  sterile  bouillon. 

The  lactose  litmus  agar  medium  is  very  useful  in  distinguishing 
typhoid  or  paratyphoid  colonies  (blue)  from  colon,  and  Streptococcus 


THE  URINE  295 

or  Staphylococcus  colonies  (pink).     The  urine  may  be  added  to  tubes 
of  melted  agar  and  then  poured. 

The  most  satisfactory  procedure  is  to  deposit  one  drop  on  a  poured  plate  and  five 
drops  on  a  second  plate.  The  surface  is  smeared  over  with  a  bent  glass  rod  first 
smearing  out  the  single  drop  and  then  going  to  the  second  plate  without  a  second 
sterilization.  Neutral  glycerine  agar  or  blood  agar  is  desirable  for  such  organisms 
as  pneumococci  or  streptococci  and,  for  the  Gonococcus,  Thalman's  medium  smeared 
over  with  a  few  drops  of  human  serum  or  Vedder's  starch  agar. 

Cystitis  from  a  colon  infection  gives  an  acid  urine;  that  caused  by  Proteus  vulgar  is 
an  alkaline  urine. 

The  old  designation  B.  termo  so  often  employed  in  connection  with  the  bacteri- 
ology of  the  urine  in  older  works  applied  to  the  proteus  group  and  M .  urea  to  ordi- 
nary staphylococci. 

The  bacillus  of  typhoid  and  the  micrococcus  of  Malta  fever  are  also 
found  in  the  urine.  This  elimination  in  urine  of  bacilli  by  typhoid 
carriers  is  of  great  importance  in  the  spread  of  the  disease. 

While  the  smegma  bacillus  in  urine  may  be  differentiated  from  the  tubercle 
bacillus  by  the  former  losing  its  red  color,  by  prolonged  decolorization  with  acid 
alcohol,  yet  it  is  chiefly  by  the  subcutaneous  inoculation  of  the  guinea-pig  that  we 
should  diagnose  genito-urinary  tuberculosis.  Inject  the  sediment  after  centrifuging. 

The  method  recommended  by  Gasis  which  depends  on  the  alkali  fast  properties 
of  the  T.  B.  has  not  given  me  satisfactory  results. 

Smegma  bacilli  are  not  disintegrated  by  antiformin  as  are  other  bacilli  than  the 
tubercle  one,  so  that  treatment  of  urinary  sediments  with  antiformin  for  finding 
tubercle  bacilli  does  not  differentiate  those  of  smegma. 

Gonococci  are  reported  from  Gram-stained  smears. 

To  culture  Gonococcus  material  the  transfer  to  culture  media  should  be  made 
almost  immediately  after  obtaining  the  material  from  the  patient.  M .  catarrhalis 
is  a  rare  finding. 

Staphylococcus  and  Streptococcus  infections  about  the  throat  as  well 
as  such  infections  in  heart  or  joint  may  show  the  presence  of  the  causa- 
tive organisms  in  the  urine.  At  times  bacterial  infections  of  the  kidney 
may  give  symptoms  of  renal  stone. 

As  it  is  much  easier  to  culture  urine  than  blood  a  bacteriological  examination  of 
the  urine  may  give  us  the  desired  information  and  the  organism  for  the  autogenous 
vaccine.  Salt  mouth  bottles  with  cotton  plugs,  when  sterilized,  make  cheap  and 
satisfactory  containers,  The  urine  should  be  plated  out  as  soon  as  possible  after 
its  passage.  As  a  rule  when  organisms  are  present  in  the  urine  they  are  in  such 
numbers  that  the  question  of  contamination  rarely  arises. 

Yeasts  and  moulds  frequently  contaminate  urine,  especially  diabetic  urine,  after 
it  has  been  passed.  Amoebae  and  flagellates  (Trichomonas  vaginalis  in  females) 
may  be  found  in  urine. 


396  URINARY  SEDIMENTS 


irlincrc  • 


Eggs  of  Schistosoma  hcematobium  (bilharziosis)  are  important  diagnostic  findings; 
these  are  terminal-spined.     Those  of  rectal  bilharziosis  are,  as  a  rule,  lateral-spined. 

In  chylous  urine  the  filarial  embryos  may  be  found.     This  exami- 
nation is  facilitated  by  centrifugalization. 

The  eggs  of  the  E.  gigas  may  be  recognized  in  urinary  sediment  by  their  pitted 
appearance. 

The  vinegar  eel  may  be  found  in  the  urine  of  females  who  have  used  vaginal 
douches  of  vinegar. 

Echinococcus  booklets,  scolices,  or  laminated  membrane  have  been  found  in  the 
urine. 

The  larval  dibothriocephalid,  Sparganum  mansoni,  has  been  reported  three  times 
in  urine  (urethra). 

Oxyuris  from  the  vagina  may  be  found  in  urine. 

Various  mites  may  be  found  in  urinary  sediment  as  the  result  of  lack  of  care 
the  washing  of  the  receptacle  and  are  entirely  accidental. 


ics 

: 


Unless  having  the  characteristics  of  the  itch  mite  and  in  a  persoi 
showing  scabies  lesions  about  the  genital  organs  the  diagnosis  of  the 
mite  as  A.  scabiei  should  not  be  made. 

Crystals  of  biliverdin  may  be  found  in  the  urinary  sediment  in  marked  jaundice. 
They  somewhat  resemble  crystals  of  tyrosin  but  are  brownish  in  color  while  those 
of  tyrosin  are  black.  Furthermore,  it  is  excessively  rare  to  find  crystals  of  leucin 
and  tyrosin  in  the  urinary  sediments,  and  in  such  diseases  as  acute  yellow  atrophy  of 
the  liver,  the  urine  should  be  concentrated  to  one-tenth  its  volume  and  the  residue 
treated  with  alcohol.  The  tyrosin  crystalline  sheaves  and  the  leucin  striated 
globules  crystallize  out  from  the  alcohol. 

URINARY  SEDIMENTS 

Turbidity  of  the  urine  is  most  often  due  either  to  bacterial  contamination,  amo 
phous  urates  (sedimentum  lateritium)  or  phosphates. 

Urates  go  into  solution  upon  heating  and  phosphates  upon  the  addition  of  a  few 
drops  of  acetic  acid. 

In  turbidity  due  to  bacteria  contaminating  the  urine  subsequent 
to  its  passage  it  is  best  to  call  for  another  sample. 

To  preserve  urinary  sediments  formalin  is  the  best  for  casts  and  epithelial  ce 
while  for  general  use  one  may  employ  a  piece  of  camphor  or  the  addition  of  o 
volume  of  saturated  borax  solution  to  four  volumes  of  urine. 

Chloroform  does  not  answer  for  sediments  as  it  does  for  urine  to  be  examined 
chemically.  To  take  up  a  sediment  insert  a  pipette  to  the  bottom  of  the  tube  with 
the  opposite  opening  closed  by  a  finger,  then  tease  the  sediment  into  the  pipet 
opening  in  the  centrifuge  tube,  by  manipulating  the  fingers. 


THE  URINE 


397 


In  a  urine  of  acid  reaction  we  may  find  the  following  unorganized  sediment: 

I.  Amorphous  sodium  or  potassium  urates.     Usually  yellowish  red.  Heat  and 
alkali  bring  about  solution. 

II.  Uric  acid.     Whetstone  crystals  of  yellowish-red  color.     Soluble  in  alkalis 


FIG.  n;o6. — Deposit  in  acid  fermentation,     a.  Fungus;  b,  amorphous  sodium  urate; 
c,  uric  acid;  d,  calcium  oxalate. 

but  not  by  heat.  Abundant  sediment  of  uric  acid  crystals  may  be  due  to  too  great 
concentration  or  too  great  acidity  of  the  urine  rather  than  to  the  so-called  uric 
acid  diathesis. 


-b 


FIG.  107. — Deposit  in  ammoniacal  fermentation,     a,  Acid  ammonium  urate,  d; 
ammonium  magnesium  phosphate;  c,  bacteria. 

III.  Calcium  oxalate.     Octahedral  crystals  or  dumb-bell  shapes  which  are  highly 
refractile.     Often  due  to  diet  (asparagus,  tomatoes,  spinach,  rhubarb,  etc.). 

IV.  Cystin  occurs  in  six-sided  crystals  which  are  soluble  in  ammonia. 


398 


EPITHELIAL  CELLS  IN  URINE 


In  a  urine  of  alkaline  reaction  we  may  expect: 

I.  Triple  phosphates  (NH4  MgPO4).     Usually  in   coffin-lid  or  fern-like  form. 
Easily  soluble  in  acetic  acid. 

II.  Calcium  phosphate  and  calcium  carbonate  which  effervesce  on  the  addition 
of  acid. 

III.  Ammonium  urate.     These  show  as  the  thorn-apple  structures. 

The  presence  of  ammonium  urate,  particularly  if  with  triple  phosphates,  denotes 
bacterial  decomposition  within  the  genito-urinary  tract  provided  the  urine  is  just 
passed.  Pus  cells  derived  from  the  site  of  inflammation  should  be  present  also. 
While  certain  bacteria  might  possibly  bring  on  chemical  changes  without  giving  rise 
to  inflammation  yet  such  a  possibility  is  so  rare  as  to  be  negligible.  In  the  presence 
of  amorphous  phosphates  one  should  always  think  of  exogenous  sources  as  vegetable 
diet  or  withdrawal  of  proteid  food  before  thinking  of  disordered  metabolism. 


FIG.  108. — Epithelium  from  different  areas  of  the  urinary  tract,  a,  Leukocyte 
(for  comparison);  b,  renal  cells;  c,  superficial  pelvic  cells;  d,  deep  pelvic  cells;  e, 
cells  from  calices;  /,  cells  from  ureter;  g,  g,  g,  g,  g,  squamous  epithelium  from  the 
bladder;  h,  h,  neck-of-bladder  cells;  i,  epithelium  from  pros ta tic  urethra;  k,  urethral 
cells;  /,  /,  scaly  epithelium;  m,  m',  cells  from  seminal  passages;  n,  compound  granule 
cells;  o,  fatty  renal  cell.  (Ogden.) 

Organized  Sediment. — An  occasional  leukocyte  may  be  found  in  the  urine  of 
healthy  people.  Any  abundance  of  leukocytes  indicates  inflammation  of  genito- 
urinary tract.  Some  workers  count  the  pus  cells  in  urine  by  the  same  technic  used 
for  the  leukocyte  count  of  the  blood.  A  urine  having  100,000  pus  cells  per  c.mm. 
will  give  as  a  result  about  0.1%  albumin. 

Leukocytes  are  found  in  abundance  at  times  in  the  urine  of  women  without 
pathological  significance.  Red  blood  cells  usually  show  as  pale  doubly  ringed 
bodies.  They  appear  in  inflammations,  particularly  stone  or  schistosome  infection, 
also  with  neoplasms  or  in  chronic  passive  congestion.  They  may  be  found  in 
conditions  where  toxins  are  being  eliminated  through  the  kidneys,  as  in  tubercu- 


THE  URINE 


399 


losis.  The  menstrual  period  of  women  must  be  kept  in  mind  in  the  examination 
of  urine  sediments. 

Epithelial  Cells. — For  morphology  of  cells  from  different  locations  see  illustration. 
It  is  almost  impossible  to  state  positively  the  origin  in  the  genito-urinary  tract  of 
certain  cells.  Very  trustworthy  evidence  however  is  finding  of  epithelial  cells  on 
casts  or  the  so-called  compound  granule  cells  (fatty  degenerated  renal  epithelium). 
Sheets  of  more  or  less  small  round  or  caudate  epithelial  cells  are  rather  significant  of 
pyelitis.  Vaginal  epithelium  resembles  that  gotten  from  scraping  the  buccal 
mucosa. 

Bladder  epithelium  resembles  vaginal  but  is  of  smaller  size,  and  ureteral  is  like 
that  of  the  pelvis  of  the  kidney  but  smaller.  Cells  from  the  region  of  the  prostate 
are  very  refractile  with  a  distinct  nucleus  and  are  oval  rather  than  round. 


FIG.  109.— Fatty  and  waxy  casts,     a,  Fatty  casts;  b,  waxy  casts.     (Greene.') 


Casts.— Of  casts  we  have  (i)  hyaline,  narrow  and  homogenous. 
They  do  not  prove  nephritis.  (2)  Epithelial  casts.  Usually  indicative 
of  nephritis  but  very  slight  inflammatory  processes  can  cause  them. 
(3)  Blood  casts.  (4)  Granular  casts.  If  coarse  granules  rather  sig- 
nificant of  chronic  nephritis.  Finely  granular  casts  do  not  seem  to 
have  any  more  significance  than  hyaline  ones.  As  a  matter  of  fact 
under  dark  ground  illumination  hyaline  casts  show  a  granular  structure. 
(5)  Waxy  casts  are  highly  refractile,  show  fissuring  of  margins  and  were 
formerly  considered  of  serious  prognostic  import  (chronic  nephritis), 


40O 


STARCHES  AND  FIBRES  IN  URINE 


but  the  present  view  is  that  they  are  only  ordinary  casts  which  have 
been  retained  in  the  renal  tubules  for  a  long  time.  Even  amyloid 
kidney  does  not  produce  any  distinctive  cast. 

Cylindroids  are  drawn-out  bodies  showing  tapering  ends,  irregularity  of  diameter 
and  longitudinal  striations. 

It  will  be  found  that  a  ^  in.  objective  gives  almost  all  the  information  required 
as  to  casts.  It  is  quicker  and  gives  more  positive  information. 

Mounting  a  sediment  in  Gram's  solution  or  tinging  it  with  the  merest  trace  of 
neutral  red  is  of  much  assistance. 


FIG.  no. — Fibres,  starch  granules,  etc.,  which  may  be  found  in  urine  sediment. 
No.  12  gives  appearance  under  microscope  of  scratches  on  old  used  glass  slides. 
No.  1 5  (a) ,  Tyroglyphus  longior,  a  mite.  No.  1 5  (b) ,  Trichomonas  vaginalis.  No.  1 6  (a) , 
Egg  of  Eustrongylus;  (6),  Echinococcus  hooklets',  (c),  Schislosoma  egg;  and  (d), 
Filaria  bancrofti  embryo. 

Starches  and  Fibres. — In  examining  urinary  sediments  it  is  important  to  be  fa- 
miliar with  the  various  textile  fibres  and  starch  grains  which  are  so  frequently  present, 
the  fibres  coming  from  the  clothing  and  the  starch  grains  from  dusting  powders. 
Wool  fibre  fragments  show  bark  or  scale-like  imbrications  and  are  round.  Cotton 
fibres  are  flattened  and  twisted,  while  linen  ones  show  a  striated  flattened  fibre  with 
frayed  segments  as  of  a  cane  stalk.  Silk  shows  a  glass-like  tube  with  mashed  in  ends. 

Corn  and  rice  grains  are  the  most  common  of  the  starch  grains  and  their  nature  is 
immediately  disclosed  by  their  blue  color  when  mounted  in  iodine. 


THE  URINE  40I 

Haematuria.— By  this  we  mean  the  presence  of  red  blood  cells  in 
the  urine,  and  the  condition  is  of  ten  recognizable  only  by  microscopic 
or  occult  blood  examinations.  It  is  better  to  separate  this  condition 
from  haemoglobinuria.  Origin  may  be  renal,  cystic,  urethral,  ureteral 
and  secondary  to  disease  or  traumatism.  It  may  also  result  from 
affections  of  structures  adjacent  to  the  genito-urinary  apparatus, 
especially  ulcerations  of  the  large  intestines  (amoebic),  or  from  disease 
of  the  female  generative  apparatus  as  uterus,  vagina  or  tubes. 

In  certain  general  diseases,  as  smallpox,  purpura,  and  leukemia  one  may  expect 
hagmaturia.  In  the  tropics  it  is  a  finding  in  yellow  fever  (asthenic  stage)  and  in 
plague  as  well  as  in  bilharziasis  and  haematochyluria  of  filariasis. 

Haemorrhage  from  the  kidneys  may  arise  from  malignant  growths,  especially 
hypernephroma,  or  from  benign  ones,  as  papilloma  of  the  pelvis. 

Haematuria  occurs  not  only  in  acute  nephritis  but  in  some  chronic 
cases.  Chronic  passive  congestion  is  associated  with  red  cells  in  the 
urine  and  renal  infarctions  from  endocarditis  may  also  bring  it  about. 

Stone  in  the  pelvis  of  the  kidney  is  an  important  cause.  In  the  bladder  we  have 
as  chief  causes  (i)  tumors,  malignant  or  innocent,  and  (2)  calculus.  In  tumors 
the  bleeding  is  not  markedly  controlled  by  rest  as  is  that  from  stone. 

In  the  urethra  gonorrhoeal  inflammation,  especially  when  near  the  neck  of  the 
bladder,  and  traumatism  may  be  associated  with  hsematuria. 

Haemoglobinuria. — The  two  diseases  one  always  thinks  of  in  con- 
nection with  haemoglobinuria  are  blackwater  fever  and  paroxysmal 
haemoglobinuria.  It  is  discussed  under  tests  for  transfusion. 

Bacteriuria. — Bacterial  infections  of  the  genito-urinary  tract  are 
associated  with  more  or  less  pyuria.  Kidney  infections  are  now 
recognized  as  most  often  from  the  blood  stream  rather  than  from 
extension  from  portions  of  the  tract  lower  down. 

The  most  important  hsematogenous  bacterial  infections  of  the  kidney  are,  in 
order  of  frequency,  colon  bacillus,  staphylococci,  streptococci,  gonococcus,  proteus, 
typhoid  and  paratyphoid.  Renal  tuberculosis  is  generally  haematogenous  rather 
than  an  ascending  infection.  Very  important  is  the  differentiation  of  the  pyuria 
of  cystitis  and  pyelitis.  Of  course  bladder  irritation  will  follow  pyelitis. 

The  use  of  two  sedimentation  glasses  will  differentiate  pus  from  the  urethra  from 
that  from  bladder  and  pelvis  of  kidney.  If  the  urine  in  the  first  glass  alone  is  turbid 
it  shows  urethral  pus.  With  cloudiness  of  the  contents  of  the  second  glass  the 
problem  is  more  difficult  because  it  is  almost  impossible  to  differentiate  a  bladder 
involvement  from  a  renal  one  by  microscopic  examination.  Cystoscopy  is  necessary. 
Of  course,  some  authorities  attach  importance  to  the  character  of  pelvis  epithelium, 
others  to  the  acid  urine  of  pyelitis  and  the  alkaline  one  of  cystitis.  Again  it  is 
26 


402  AMBARD 's  INDEX  OF  UREA  EXCRETION 

often  noted  that  the  albumin  content  of  the  urine  of  pyelitis  is  far  greater  than  that 
from  cystitis.     This  is  true  for  pyelonephritis  but  does  not  hold  for  simple  pyelitis. 

Pyelitis,  usually  a  colon  infection,  must  always  be  thought  of  in 
vague  disorders  of  children  and  pregnant  women. 

Renal  stone  and  renal  tuberculosis  often  give  similar  symptoms  but  the  X-ray 
and  animal  inoculation  test  should  differentiate. 


DETERMINATION  OF  EFFICIENCY  OF  RENAL  FUNCTIONING 

At  present  we  are  paying  great  attention  to  laboratory  tests  which 
give  us  an  idea  of  the  activity  of  nitrogen  metabolism  and  efficiency 
of  the  renal  functions.  Probably  the  most  reliable  single  test  is  the 
phenolsulphonephthalein  or  "red"  test.  This  is  given  in  the  appendix. 
For  alveolar  CO2  content  see  Acidosis. 

Determination  of  the  ammonia  output  in  the  urine  is  also  of  value  in  conditions 
of  acidosis.  In  acidosis  connected  with  diabetes  we  expect  a  great  increase  in  the 
urinary  ammonia  to  neutralize  acetone  bodies  as  is  also  true  of  degenerations  invol- 
ving the  parenchymatous  liver  cells,  when  the  urea  function  is  interfered  with. 
In  the  acidosis  of  chronic  nephritis,  however,  we  may  have  a  deficiency  in  the  ammo- 
nia output  in  the  urine.  Simple  tests  for  this  determination  will  be  found  in  the 
appendix. 

Great  attention  is  being  given  to  the  nonprotein  nitrogen  of  the  blood  as  well 
as  to  blood  urea.  See  appendix. 

The  study  of  nitrogen  metabolism  is  best  undertaken  with  a  patient 
on  a  known  nitrogen  value  diet  and  the  most  accurate  determination 
is  along  the  lines  of  the  Ambard  index  of  urea  excretion. 

Ambard  Index. — McLean  has  worked  out  an  index  of  urea  excretion  based  on 
the  Ambard  variables  of  (i)  concentration  of  urea  in  the  blood.  (2)  Concentration 
of  urea  in  the  urine.  (3)  Rate  of  urinary  excretion  and  (4)  weight  of  the  individual. 
McLean's  formula  is  as  follows: 

_  £. 

=  Index  of  urea  excretion. 


D  =  Grams  urea  excreted  in  twenty-four  hours. 

C  =  Grams  urea  per  liter  urine. 
Ur  =  Grams  urea  per  liter  blood. 
Wt  =  Eody  weight,  individual  in  kilograms. 

One  hundred  is  accepted  as  a  typical  normal  finding,  and  findings  down  to  80 
as  within  normal  limits.     In  deficiency  of  renal  function  the  index  falls  below  50 
and  in  cases  of  marked  deficiency  may  fall  below  10,  and  in  terminal  stages 
index  may  approximate  i.     Such  cases  practically  show  an  absence  or  only  a  trace 
phenolsulphonephthalein  excretion. 


sthe 

.. 


THE  URINE  403 

For  determination  of  urea  excretion  the  patient  drinks  200  c.c.  water  and  one- 
half  hour  later  empties  his  bladder.  Commencing  at  this  we  wait  seventy-two 
minutes  (one-twentieth  of  twenty-four  hours)  and  then  collect  the  urine  for  urea 
determination. 

The  blood  for  urea  determination  should  be  taken  midway  in  the  period  of  col- 
lection of  the  urine — thirty-six  minutes  after  the  bladder  is  voided. 

Salt  Retention. — As  a  test  for  salt  retention  Schlayer  advises  giving 
10  grams  of  NaCl  to  a  patient  having  a  salt  equilibrium  as  the  result 
of  a  known  diet  continued  over  several  days.  This  added  salt  should 
be  eliminated  in  the  urine  within  twenty-four  hours  or  certainly  by 
forty-eight  hours.  Monakow  in  a  similar  way  gives  20  grams  of  urea 
at  one  dose  and  this  added  urea  should  be  eliminated  in  twenty-four 
to  forty-eight  hours  to  show  normal  nitrogenous  output. 

A  case  of  nephritis  may  show  a  good  urea  elimination  but  salt  retention. 

Diminished  capacity  to  eliminate  salt  is  a  rather  constant  finding  in  all  types  of 
chronic  nephritis. 

Frothingham  considers  elimination  of  8.5  grams  of  the  10  grams  administered  as 
normal,  above  4.5  grams  as  slight  retention  and  below  4.5  grams  as  marked  retention. 

Lactose  Excretion. — Intravenous  injection  of  20  grams  lactose  in  20 
c.c.  distilled  water.  The  solution  should  be  pasteurized  at  8o°C.  for 
three  hours  on  three  successive  days.  The  urine  is  collected  at  one- 
to  two-hour  intervals  and  tested  with  Nylander's  test  for  sugar  until 
you  get  negative  reaction.  Normally  all  lactose  is  excreted  in  four 
or  five  hours. 


404 


THE  URINE  IN  RENAL  DISEASE 


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1 


CHAPTER  XXVIII 
THE  FAECES 

IT  is  advisable  to  examine  a  stool  macroscopically  before  taking  up 
the  microscopical  examination.  The  mucus  shreds  or  casts  of  the 
bowel  in  mucous  colitis  or  membranous  enteritis  may  give  the  diag- 
nosis of  obscure  abdominal  pain.  Pus  in  stools  may  often  be  noted 
without  the  aid  of  the  microscope. 

The  normal  stool  is  sausage  shaped  and  soft.  Neither  the  special  form  of  scybal- 
ous  masses  called  sheep  pellets  nor  the  pencil-like  nor  the  tape-like  excrement  prove 
the  existence  of  stricture  of  the  intestinal  lumen  although  suggestive  of  such  a  con- 
dition. The  mucus  of  bacillary  dysentery  is  opaque  and  grayish  from  the  great 
number  of  pus  and  phagocytic  cells.  It  is  well  to  remember  that  Charcot  Leyden 
crystals,  which  are  practically  always  absent  from  bacillary  dysentery  stools,  are 
not  infrequent  findings  in  the  amoebae  containing  stools;  of  course,  these  crystals 
appear  in  other  intestinal  parasite  infections. 

In  obstruction  of  the  common  bile  duct  we  have  acholic,  whitish,  foul-smelling 
stools.  If  the  putty  color  be  due  to  bacterial  change  exposure  to  the  air  will  restore 
the  brownish  tinge. 

Sprue  stools  are  white-wash  to  putty  colored,  pultaceous,  and  filled  with  air 
bubbles.  The  amount  is  excessive. 

Fatty  stools  are  best  examined  microscopically. 

As  so  many  solid  masses  resemble  gallstones  it  is  well  to  dissolve  the  suspected 
mass  in  hot  alcohol  and  examine  for  cholesterin  crystals  upon  evaporation  of  the 
alcohol. 

If  the  faecal  examination  is  to  be  made  for  the  diagnosis  of  amoebae, 
in  a  case  where  the  characteristic  mucus  stools  are  not  present,  or  to 
verify  the  existence  of  flagellates,  it  is  best  to  give  a  dose  of  salts  early 
in  the  morning  and  examine  the  liquid  stools  which  follow  such  treat- 
ment. This  treatment  is  satisfactory  for  examination  for  intestinal 
parasites  or  ova. 

A  very  practical  way  of  obtaining  amoebae  is  to  pass  a  rectal  tube  or  a  piece  of 
drainage  tube  with  fenestrations  into  the  bowel,  and  amoebae  may  be  found  in  the 
mucus  filling  the  perforations  in  the  tube.  Walker  advises  against  the  use  of  salts 
in  examinations  for  amoebae. 

If  the  purpose  of  the  examination  is  to  determine  the  digestive  power 
of  the  alimentary  tract  for  proteids,  carbohydrates,  or  fats,  it  is  best 
to  use  a  test  diet,  as  that  of  Schmidt  and  Strasburger. 

405 


406 


SCHMIDT  TEST  DIET 


TTiT^if* 


Prior  to  using  this  test  diet,  one  should  familiarize  himself  with  the  macroscopic 
and  microscopic  appearances  resulting  from  such  a  diet  in  a  normal  person;  informa- 
tion is  then  at  hand  to  judge  of  variations  from  the  normal.  The  examination  of 
the  faeces  of  persons,  on  ordinary  and  specifically  undetermined  articles  of  diet,  is 
very  unsatisfactory  when  the  state  of  digestion  of  muscle  fibers  and  the  question  of 
fat  digestion  are  at  issue. 

In  examining  the  faeces  of  the  normal  person  and  likewise  with  the  patient,  wait 
until  the  second  or  third  day  so  that  the  faeces  of  previous  diets  may  have  passed  out. 
A  charcoal  powder  taken  before  commencing  the  diet  serves  as  an  indicator. 

Diet:  breakfast,  7  A.  M.,  bowl  of  oatmeal  gruel  (40  grams  oatmeal,  10  grams 
butter,  200  c.c.  milk,  300  c.c.  water).  Also  one  very  soft-boiled  egg  (one  minute) 
and  50  grams  zwieback.  In  the  forenoon,  500  c.c.  of  milk. 

For  dinner,  2  o'clock,  chopped  beef  broiled  very  rare  (125  grams  with  20  grams 


FIG.  in. — Microscopical  constituents  of  faeces,  (v.  Jaksch.)  a,  Muscle  fibres; 
6,  connective  tissue;  c,  epithelium;  d,  leukocytes;  e,  spiral  cells;  /,  g,  h,  i,  various 
vegetable  cells;  k,  "triple  phosphate"  crystals;  /,  woody  vegetable  cells;  the  whole 
interspersed  with  innumerable  microorganisms  of  various  kinds. 

butter  poured  over  it).  Also  a  potato  puree  (200  grams  mashed  potato,  50  grams 
milk,  10  grams  butter).  Also  J^  liter  of  milk  and  50  grams  zwieback. 

For  supper,  7  o'clock,  the  same  articles  as  for  breakfast. 

This  detailed  diet  may  be  varied  to  suit  circumstances  as  regards  interchanging 
meals.  Furthermore,  the  milk  may  be  taken  in  the  form  of  tea  or  cocoa  or  cooked 
with  the  other  food.  Even  a  small  amount  of  wine  may  be  permitted.  The  diet 
taken,  however,  should  absolutely  conform  to  the  following  requirements:  i.  the 
taking  of  Y±  pound  chopped  beef,  a  portion  of  which  should  be  half  raw;  2.  the  milk 
taken  should  amount  to  about  a  quart;  3.  about  4  ounces  of  bread  or  toast  and  from 
4  to  8  ounces  of  potato  puree  should  be  eaten  daily. 

The  detailed  diet  contains  about  no  grams  albumin,  105  grams  fat  and  200  grams 
carbohydrates  with  a  fuel  value  of  2247  calories. 

The  stool  is  best  collected  in  quart  fruit  jars  and  examined  as  soon  after  evacua- 
tion as  possible.  The  wooden  spatula  like  tongue  depressors  are  well  adapted  to 
handling  the  specimen. 


THE 


407 


Having  familiarized  one's  self  with  the  degree  of  digestion  of  muscle,  starch, 
and  fat  in  a  normal  person,  we  are  in  a  position  to  judge  of  the  state  of  assimilation 
in  a  patient. 

The  first  part  of  the  test  is  the  macroscopical  one.  For  this  grind  up  a  faecal 
mass  of  y%  to  i  inch  diameter  in  a  mortar,  gradually  adding  water  until  it  has  the 
consistence  of  a  broth.  About  %  c.c.  of  this  emulsion  should  now  be  squeezed 
out  between  two  slides  and  studied  against  a  dark  surface  and  then  when  held  up 
to  the  light.  The  normal  stool  gives  a  rather  uniform  brownish  homogeneous  layer. 
Connective-tissue  remnants  (indicative  of  gastric  derangement)  show  as  whitish 
fibers.  Undigested  muscle  tissue  remnants  as  reddish-brown  splotches.  Fat 
particles  as  whitish-yellow  clumps.  Potato  remnants  appear  like  sago  grains  and 
mash  out  easily  like  mucus.  Mucus  is  best  noted  in  the  faecal  mass  before  making 
the  emulsion.  In  the  microscopical  test  of  this  emulsion: 

We  judge  of  muscle  digestion  by  the  intactness  of  the  stria tions. 
If  a  muscle  remnant  is  only  a  homogeneous  yellowish  particle,  it  shows 
satisfactory  digestion.  If  it  is  rectangular,  with  well-defined  cross 
striations,  it  shows  poor  digestion  for  meat  (Azotorrhoea).  A  loopful 
of  faeces  should  be  smeared  into  a  drop  of  Gram's  solution  for  starch- 
digestion  determination.  Normally  there  should  be  no  blue-staining 
starch  granules. 

Soaps  are  gnarled  bodies  everted  like  the  pinna  of  an  ear,  while  soap  crystals 
are  comparatively  coarse  and  do  not  melt  on  application  of  gentle  heat  as  do  the 
more  delicate  fatty  acid  crystals.  Neutral  fat  is  in  round  or  irregular  globules.  The 
best  stain  for  fat  is  Sudan  in  (saturated  solution  of  Sudan  in  in  equal  parts  of 
70%  alcohol  and  acetone). 

Mix  up  the  f ssces  with  dilute  alcohol  (50  to  70%)  and  then  add  a  drop  of  the  above 
solution  and  apply  a  cover-glass  quickly.  The  fat  globules  show  as  orange  or 
golden  yellow  bodies. 

By  rubbing  up  a  small  portion  of  the  faeces  in  36%  acetic  acid,  applying  a  cover- 
glass  and  heating  over  a  flame  until  the  preparation  shows  bubbles,  we  convert  the 
soaps  and  other  fat  combinations  into  free  fatty  acids  which  show  as  more  or  less 
numerous  highly  refractile  bodies  showing  a  crystalline  structure  as  the  prepara- 
tion cools.  By  practice  one  learns  the  amount  of  such  globules  to  expect  with  differ- 
ent fat  contents  in  stools. 

Steatorrhcea,  or  the  presence  of  fat  in  abnormal  quantities  in  the 
faeces,  is  shown  by  the  pale,  bulky,  greasy  stools  as  well  as  in  the  micro- 
scopical examination. 

Average  for  normals  in  i  gram  dried  faeces: 

Total  fat,  225  mg.  (22.5%) 

Total  fatty  acid,  86  mg.  (37-9%  of  a11  fat) 

Total  soap,  74  •  7  mg.  (33 . 4%  °f  aU  fat) 

Total  neutral  fat,  64 . 4  mg.  (28 . 5  %  of  all  fat) 


408  BILE  IN  FAECES 

In  normal  cases  the  only  fat  elements  recognizable  are  yellow  calcium  or  color- 
less soaps.  In  sprue  from  25  to  30%  of  the  fat  ingested  appears  in  the  stool  while 
the  stool  of  pellagra  shows  only  about  5%  which  is  the  normal  figure. 

As  quantity  of  fat  increases  (as  say  500  to  600  mg.)  droplets  of  neutral  fat  appear 
with  or  without  increase  in  number  of  soap  masses.  Also  needles  and  splinters 
of  fatty  acid  and  soaps  appear.  Much  connective-tissue  debris  shows  defect  in 
gastric  digestion,  as  only  the  stomach  digests  connective  tissue. 

A  test  for  activity  of  fermentation  should  be  made  by  using  a  Schmidt 
apparatus. 

A  distinct  evolution  of  gas  in  twelve  hours  shows  starch  digestion  defect.  Such 
faeces  are  acid.  A  delayed  production  of  gas  (after  twenty-four  hours)  shows  albu- 
min' decomposition.  Such  faeces  show  an  alkaline  reaction.  The  apparatus  is 
shown  in  Fig.  7.  Into  a  stocky  salt  mouth  bottle  we  put  approximately  5  grams 
of  faeces  which  have  been  rubbed  up  into  an  emulsion  with  water  and  fill  the  bottle 
with  water.  The  remaining  portion  of  the  apparatus  consists  of  a  test-tube  or  a 
graduated  cylinder  fitted  with  a  doubly  perforated  rubber  stopper.  One  U-shaped 
glass  tube  passing  through  this  stopper  connects  with  a  second  test-tube.  This  tube 
serves  as  a  receptacle  for  any  water  which  may  come  over  from  the  water-filled  tube 
or  graduated  cylinder  and  has  an  opening  punched  out  of  the  bottom  of  the  test- 
tube.  The  other  opening  in  the  twice  perforated  cork  admits  a  straight  tube  which 
connects  with  a  large  rubber  stopper  which  fits  into  the  bottle  for  the  faeces.  To 
prepare,  fill  the  graduated  cylinder,  then  push  in  the  doubly  perforated  cork  which 
is  connected  with  the  side  receiving  tube  and  the  large  rubber  cork.  This  latter  is 
then  pushed  down  to  fit  tightly  into  the  bottle  filled  full  with  the  faeces  emulsion. 

In  addition  to  the  faeces  examination  we  should  check  the  results  from  the  test 
diet  with  indican  and  nitrogen  partition  determinations  of  the  twenty-four-hour 
urine  specimen — the  ratio  of  ammonia  nitrogen  to  total  nitrogen  indicating  the  func- 
tional power  of  liver  and  the  indican  the  question  of  stasis  in  lower  part  of  small 
intestine. 


The  most  satisfactory  test  for  bile  in  the  faeces  is  to  emulsify  a  small 
particle  of  faeces  in  a  saturated  aqueous  solution  of  bichloride  of  mercury, 
preferably  with  a  wooden  toothpick,  on  a  concave  glass  slide.  After 
one  or  more  hours  hydrobilirubin-containing  faeces  show  a  salmon  pink 
color  and  bilirubin  ones  a  green  color.  One  should  familiarize  himself 
with  these  reactions  in  normal  cases.  The  examination  of  faeces  for 
bile  is  less  certain  than  duodenal  fluid  examination. 

In  examining  a  liquid  stool  after  salts,  it  is  well  to  color  the  drop  of  faeces,  which 
is  to  be  covered  with  the  cover-glass,  with  a  small  loopful  of  %%  solution  of  neutral 
red.  If  diluting  fluid  is  used,  it  should  be  salt  solution,  and  not  water.  The  neutral 
red  tinges  the  granules  of  the  endoplasm  of  amoebae  and  flagellates  a  very  striking 
rose  pink  color,  thus  differentiating  them  from  vegetable  cells  or  body  cells. 

Whether  examining  the  thin  faeces  or  the  mucus  particle,  it  is  well  to  reserve 
report  on  amoebae  or  flagellates  until  motion  is  observed.  Encysted  protozoa  are 


THE  FAECES  409 

difficult  to  diagnose.     An  experienced  examiner  easily  recognizes  the  four  or  eight 
nucleated  cysts  when  the  material  is  mounted  in  Gram's  iodine  solution. 

When  a  smear  preparation  is  desired,  we  may  smear  out  a  fragment  of  mucus 
and  stain  by  Romanowsky's  or  Gram's  method.  The  character  of  the  bacteria 
present  appears  to  be  of  diagnostic  value— especially  in  the  case  of  infants  and  young 
children.  Beautiful  preparations  may  be  made  by  mixing  the  faeces  with  water,  then 
centrifuging  for  one  minute.  This  throws  down  vegetable  debris  and  crystals.  Now 
decant  the  supernatant  fluid,  which  holds  the  bacteria  in  suspension,  and  add  an 
equal  amount  of  alcohol.  Again  centrifuge,  decant,  and  smear  out  and  examine 
the  bacterial  sediment. 

Simply  taking  a  small  mass  of  faeces  and  emulsifying  it  with  a  wooden 
toothpick  on  a  concave  slide  in  70%  alcohol— then,  after  the  sedi- 
ment settles,  taking  up  a  loopful  with  platinum  loop  from  the  surface 
and  smearing  out,  gives  a  very  satisfactory  smear.  Gram's  method, 
with  dilute  carbol  fuchsin  counterstaining,  gives  the  best  picture. 

The  Boas-Oppler  bacillus  may  be  found  in  the  stools  in  this  way.  Normally, 
a  Gram-stained  stool  shows  a  great  preponderance  of  Gram-negative  bacilli  and  such 
a  finding  in  a  measure  excludes  cancer  of  the  stomach.  Organisms  which  are  Gram- 
positive  as  well  as  the  Boas-Oppler  bacillus  are,  i.  Lactic  acid  bacilli — these  show 
Gram- negative  areas  in  the  slender  bacilli.  2.  A  type  of  bacillus  similar  in  size  to  the 
colon  bacillus  but  Gram-positive  and  noncultivable  (found  in  acid  stools).  3.  Bacilli 
of  the  B.  subtilis  type. 

It  is  very  important  to  examine  the  faeces  for  T.  B.  With  children  a  diagnosis 
of  tuberculosis  may  be  made  in  this  way  when  the  sputum  cannot  be  obtained, 
the  pulmonary  secretion  being  swallowed.  The  preparation  on  the  concave  slide 
as  described  above  should  be  stained  for  T.  B.  Ulcerations  in  intestinal  T.  B.  may 
show  very  numerous  bacilli. 

To  culture  for  typhoid,  dysentery,  cholera,  or  other  bacteria,  take  up  the  material 
in  a  tube  of  sterile  bouillion  and  smear  it  out  with  a  swab  over  a  lactose  litmus  agar 
plate  or  an  Endo  or  Conradi-Drigalski  plate.  Before  streaking  the  plates  they 
should  be  very  dry  on  the  surface.  This  can  be  best  done  by  pouring  into  a  plate 
with  a  circular  piece  of  filter-paper  in  the  lid  and  placing  in  the  incubator  for  one-half 
hour  to  dry.  The  filter-paper  absorbs  the  moisture.  Then  inoculate  the  surface  of 
the  plate  with  the  faecal  material.  Selective  cholera  plating  media  are  strongly 
alkaline. 

In  summer  complaints  of  infants  and  children  the  organisms  con- 
cerned are  as  a  rule  related  to  various  dysentery  strains  of  bacilli. 
Kendall  in  293  stool  examinations  found  the  gas  bacillus  (B.  Grog, 
capsul.)  in  22  cases.  The  gas  bacillus  produces  intestinal  disorders 
which  are  not  benefited  by  lactose  but  by  buttermilk  (lactic  acid  bac- 
teria). For  diagnosis,  a  loopful  of  the  faeces  is  emulsified  in  a  tube  of 
sterile  milk  or  litmus  milk.  The  emulsion  is  heated  to  8o°C.  and  held 
at  this  temperature  for  twenty  minutes.  After  incubation  for  eighteen 


410  GALL-STONES 

to  twenty-four  hours,  preferably  anaerobically,  we  get  (i)  a  shreddy 
disruption  of  the  casein,  (2)  the  smell  of  rancid  butter  and  (3)  fully  80% 
of  the  casein  is  dissolved.  Smears  show  short  thick  Gram-positive  rods 
with  slightly  rounded  ends.  B.  subtilis  is  sometimes  found  but  does 
not  give  a  rancid  odor  nor  the  strong  disruption  of  the  clot. 

It  was  until  recently  thought  that  Cammidge's  reaction  (urine)  when  associated 
with  azotorrhoea  and  steatorrhcea  made  for  a  diagnosis  of  chronic  pancreatitis. 
At  present  very  little  importance  is  attached  to  the  Cammidge  reaction. 

Loss  of  weight,  anaemia,  diarrhoea  and  pains  in  the  upper  abdomen 
are  important  indications  of  pancreatic  trouble.  As  chronic  pan- 
creatitis is  often  associated  with  cholelithiasis  jaundice  is  frequently 
present.  Glycosuria  is  not  often  present.  While  functional  tests 
are  important  they  do  not  make  for  a  sure  diagnosis.  At  present  we 
examine  the  duodenal  fluid  for  presence  of  pancreatic  ferments. 

Miiller's  method  for  pancreatic  functioning  determination  is  to  give  a  calomel 
purge  two  hours  after  a  meal.  A  little  of  the  liquid  stool  is  smeared  on  the  surface 
of  blood-serum  and  the  tube  incubated  at  6o°C.  (paraffin  oven).  If  the  surface  is 
smooth,  no  trypsin  was  present;  if  dotted  with  spots  of  digestion  liquefaction,  it 
shows  that  the  pancreatic  secretion  is  present. 

In  Schmidt's  nucleus  test  small  cubes  of  beef  are  hardened  in  absolute  alcohol 
and  then  tied  up  in  tiny  silk  bags.  These  are  recovered  from  the  faeces  and  sec- 
tioned. Complete  preservation  of  nuclei  indicates  a  total  absence  of  pancreatic 
functioning  provided  the  passage  of  the  tissue  be  not  too  rapid  as  by  diarrhoea. 

In  the  microscopic  examination,  epithelial  cells  are  generally  more  or  less  disinte- 
grated. In  the  mucus  of  bacillary  dysenteric  stools,  however,  large  intact  phago- 
cytic  cells  are  frequent,  which  may  be  mistaken  for  encysted  amoebae. 

Triple  phosphate  crystals  are  frequently  observed  in  faeces,  as  may  also  be  crys- 
tals of  various  calcium  salts.  Charcot-Leyden  crystals  are  rather  indicative  of 
helminthiases. 

Various  flagellates,  and  in  particular  Lamblia,  may  be  responsible  for  diarrhceal 
conditions  which  may  cause  rather  serious  symptoms. 

Balantidium  coli  has  been  reported  several  times  as  the  cause  of  dysenteric  con- 
ditions. Coccidiadea  are  found  in  the  faeces. 

Isospora  was  not  an  infrequent  finding  in  the  stools  of  the  soldiers  at  Gallipoli. 

Gall-stones  are  usually  recognized  by  their  facetted  appearance. 
The  stool  should  be  examined  for  two  weeks  following  an  attack  of  hep- 
atic colic  and  the  faeces  should  be  rubbed  up  in  water  and  passed  through 
a  seive.  A  concentric  arrangement  of  layers  is  usually  noted  on  frac- 
turing a  gall-stone.  For  identification  dry  and  pulverize  the  stone 
and  treat  with  alcohol  and  ether.  This  dissolves  the  cholesterin  and 
upon  evaporation  the  rhombic  crystals  separate  out  and  may  be  recog- 


THE  FAECES 

nized  microscopically.  The  residue  may  be  extracted  with  cold  dilute 
KOH  solution.  This  extracts  bilirubin  which  may  be  recognized  by 
Gmelin's  test.  Pseudo-gall-stones  are  usually  masses  of  fats,  soaps  or 
vegetable  material. 

At  times  enteroliths  are  mistaken  for  gall-stone.  These  are  shells  of  inorganic 
salts  covering  inspissated  masses  of  faeces  or  seeds,  etc. 

It  is  in  the  faeces  we  examine  either  for  the  parasites  or  for  their  ova 
in  connection  with  practically  all  the  flukes,  except  the  lung  fluke  and 
the  bladder  fluke;  for  intestinal  taeniases  and  for  practically  all  the 
round  worms,  except  the  filarial  ones. 

Bass  has  recommended  that  faeces  which  have  been  made  fluid  be  centrifuged 
and  the  supernatant  fluid  containing  vegetable  debris  poured  off.  The  sediment 
contains  hookworm  eggs.  Then  pour  on  sediment  a  calcium  chloride  solution  of 
sp.  gr.  1050.  Again  centrifuge  and  decant.  Next  add  calcium  chloride  solution 
of  a  sp.  gr.  of  1250  and  centrifuge.  This  brings  to  the  surface  the  hookworm  eggs 
which  may  be  pipetted  off.  As  a  rule,  the  finding  of  hookworm  eggs  is  very  easy 
without  such  a  technic.  The  eggs  of  Trichostrongylus  greatly  resemble  those  of 
hookworm  but  are  larger,  73  to  gi/i  long.  In  perfectly  fresh  faeces  Strongyloides  are 
present  as  worm-like  embryos  while  hookworm  gives  only  two  to  four  segment  eggs. 

In  the  tropics,  the  examination  of  the  faeces  vastly  exceeds  in  value 
that  of  urine  and  is  possibly  more  important  than  blood  examinations. 

The  larvae  of  various  insects  may  at  times  be  detected  in  the  stools,  as  well  as 
certain  acarines  (cheese  mites,  etc.). 

The  test  for  occult  blood  is  indicated  in  helminthiases  as  well  as  in  the  conditions 
for  which  it  is  usually  tested. 


CHAPTER  XXIX 
BLOOD  CULTURES  AND  BLOOD  PARASITES 

CLINICALLY,  the  most  important  examinations  of  the  blood  for 
parasites  is  for  the  presence  of  various  bacterial  infections  and  for 
certain  blood  protozoa  and  also  filarial  embryos. 

The  modern  method  of  culturing  blood,  especially  for  the  detection  of  typhoid 
or  paratyphoid  bacilli,  is  by  the  use  of  the  bile  media  of  Conradi.  Test-tubes  are 
filled  with  7  to  10  c.c.  of  i%  peptone  ox  bile,  or  ox  bile  alone,  and  the  medium  is 
sterilized  in  the  autoclave.  It  is  good  practice  to  place  the  syringe  in  a  plugged 
test-tube  containing  salt  solution,  with  the  needle  unscrewed.  After  autoclaving, 
the  sterile  syringe  can  be  taken  to  the  bedside  in  the  test-tube.  Using  a  wide  test- 
tube,  a  forceps  can  be  sterilized  at  the  same  time  and  used  to  attach  the  needle 
to  the  barrel  of  the  syringe. 

By  using  a  piece  of  glass  tubing  into  which  the  needle  is  inserted  we  may  sterilize 
the  syringe  easily  in  the  test-tube.  The  glass  tubing  prevents  the  steel  needle  from 
coming  in  contact  with  the  glass  of  the  test-tube  and  so  prevents  cracking  the  test- 
tube. 

The  skin  should  be  scrubbed  gently  with  green-soap  solution  and  water  for  about 
three  minutes.  The  skin  of  the  area  to  be  punctured  should  then  be  sterilized  by 
the  gentle  application  of  Harrington's  solution  (not  scrubbed)  for  one-half  minute, 
and  should  then  be  washed  with  sterile  water.  It  appears  to  be  safe  to  simply 
scrub  the  area  with  70%  alcohol  for  one  or  two  minutes.  Applications  of  pure 
carbolic  acid  on  a  gauze  wad  for  a  few  seconds  followed  by  neutralization  with  70% 
alcohol  gives  satisfactory  sterilization.  The  present  method  of  sterilizing  skin 
for  taking  blood  or  inoculating  vaccines  is  simply  to  smear  the  site  of  entrance  for 
the  needle  rather  heavily  with  tincture  of  iodine.  .A  tourniquet  is  now  applied  to 
distend  the  vein,  and  the  needle,  beveled  side  up,  is  inserted  in  the  direction  of  the 
venous  flow.  Withdrawing  5  to  10  c.c.  of  blood,  we  loosen  the  tourniquet  (otherwise 
the  blood  may  flow  from  the  puncture)  then  withdraw  the  needle,  and  force  out 
about  ^  c.c.  into  the  first  bile  tube,  about  i  c.c.  into  the  second,  and  2  or  3  c.c. 
into  the  third.  It  is  well  to  reserve  some  of  the  blood  for  Widal  tests. 

The  bile  tubes  are  now  incubated  for  ten  to  twelve  hours  and  then  transfers 
are  made  to  bouillon  tubes.  These  bouillon  tubes  can  be  used  in  six  to  eight  hours 
for  testing  the  organism  against  knawn  typhoid  or  paratyphoid  sera.  Test-tubes 
containing  10  c.c.  of  ordinary  bouillon  with  i%  of  sodium  citrate  are  as  satisfactory 
as  bile  media. 

Some  prefer  a  2%  sodium  glycocholate  in  bouillon  while  others  use  a  2%  solution 
of  ammonium  oxalate  in  bouillon  for  blood  cultures. 

Some  prefer  to  streak  plates  of  lactose  litmus  agar  with  material  from  the  bile 

412 


BLOOD  CULTURES  AND  BLOOD  PARASITES         413 

tubes  instead  of  inoculating  the  bouillon  tubes.  Contamination  with  staphylococci 
or  the  presence  of  staphylococci,  streptococci,  or  plague  bacilli  in  septicsemic  con- 
ditions show  easily  accessible  colonies. 

Schotmuller  adds  i  to  3  c.c.  of  blood  to  liquefied  agar  at  45°C.,  and  after  mixing 
pours  into  plates.  The  standard  method  formerly  was  to  add  the  blood  to  an 
excess  of  bouillon  (i  to  5  c.c.  of  blood  to  100  c.c.  or  more  of  bouillon). 

The  method  of  culturing  blood  we  now  follow  in  our  laboratory  is 
the  following: 

A  stout  hypodermic  needle  is  attached  to  about  6  inches  of  rubber  tubing  which  in 
turn  is  pushed  over  a  downward  bent  glass  tube  which  passes  through  a  doubly 
perforated  rubber  stopper.  A  second  glass  tube,  which  also  passes  through  the  stop- 
per, is  bent  upward  to  be  attached  to  a  second  piece  of  rubber  tubing  for  use  in  suc- 
tion by  the  mouth.  The  glass  tubes  project  about  Y^  inch  below  the  undersurface 
of  the  rubber  stopper  and  above  are  about  2%  inches  including  the  bent  arm.  This 
system  of  tubing  and  stopper  is  readily  sterilized  by  boiling  in  a  pan  or  instrument 
sterilizer.  As  a  receptacle  for  the  blood  we  employ  Erlenmeyer  flasks  of  100  c.c. 
capacity,  containing  10  to  25  c.c.  of  salt  solution  with  i  or  2%  of  sodium  citrate,  for 
prevention  of  coagulation.  These  citrated  salt  solution  flasks  are  plugged  with  cot- 
ton, sterilized  and  kept  on  hand  ready  for  immediate  use,  so  that  we  only  have  to 
sterilize  the  stopper  and  tubing  by  boiling  and  flame  the  neck  of  the  flask  when  re- 
moving the  cotton  plug  to  insert  the  stopper  of  the  system.  By  suction  we  can  take 
any  amount  of  blood  desired.  I  usually  count  the  drops  of  blood  as  they  fall  into 
the  citrated  salt  solution  allowing  16  drops  to  the  cubic  centimeter.  In  this  way  we 
may  take  from  10  to  25  c.c.  of  blood  at  the  bedside  and  then  later  on  in  the  laboratory, 
when  it  is  convenient,  inoculate  various  media  from  the  flask.  For  plates  add  2  or  3 
c.c.  of  this  citrated  blood  to  6  or  8  c.c.  of  melted  agar  at  45°C.  The  blood  mixture 
can  also  be  added  to  various  sugar  bouillons  for  fermentation  reaction.  Finally  we 
place  the  receiving  flask  in  the  incubator  and  culture  it  as  well  as  the  other  media. 

Of  course  in  inoculating  the  various  plating  or  sugar  tubes  from  the  flask  there  is 
some  liability  to  contamination.  This  may  be  avoided  by  removing  with  a  sterile 
pipette  10  to  20  c.c.  from  the  flask  containing  the  citrated  blood  to  carry  out  the 
inoculations  instead  of  pouring  out  directly  from  the  flask. 

A  very  useful  procedure  in  the  isolation  of  streptococci,  pneumo- 
cocci,  plague  and  anthrax  bacilli  is  to  inject  i  to  2  c.c.  of  blood  into 
suitable  animals.  When  infecting  mice  use  only  about  0.2  c.c.  sub- 
cutaneously  at  root  of  tail  or,  more  certain  of  results,  the  injection  of 
about  i  c.c.  of  the  blood  intraperitoneally.  Streptococci,  even  from 
virulent  human  infections,  are  uncertain  in  their  action  on  animals 
so  that  the  failure  to  produce  septicaemia  in  the  mouse  does  not  neces- 
sarily indicate  that  the  organism  is  of  slight  virulence. 

By  using  the  bile  media,  we  can  take  the  blood  from  the  ear  in  typhoid  cases, 
if  preferred.  Then  if  chance  staphylococcic  contamination  occurs,  such  colonies 
are  readily  differentiated  from  typhoid  ones  by  the  pink  color  on  lactose  litmus 


414  BACTERIAEMIA 


ITS  be 


agar.  For  culturing  blood  in  septicaemic  conditions,  the  blood  should  always 
drawn  from  the  vein  and  cultured  either  by  mixing  i  to  2  c.c.  with  melted  agar 
and  then  pouring  plates  or  by  transferring  to  bouillon  in  excess  (at  least  ten  times 
as  much  bouillon  as  blood)  and  after  eighteen  to  twenty-four  hours'  incubation 
plating  out.  For  Streptococcus  and  Pneumococcus  blood  agar  plates  are  to  be  pre- 
ferred, the  Pneumococcus  giving  green  colonies  with  only  a  suggestion  of  haemolysis 
while  the  Streptococcus  gives  an  opaque  colony  with  a  distinct  haemolytic  zone 
surrounding  it.  We  rarely  culture  blood  anaerobically  as  the  important  patho- 
genic anaerobes  (tetanus,  gas  gangrene  and  malignant  oedema)  do  not  tend  to 
invade  the  blood  stream  during  life.  Recently  a  case  has  been  reported  where  the 
gas  bacillus  was  isolated  from  the  blood  of  a  soldier  with  gas  gangrene.  There  is 
an  anaerobic  streptococcus  S.  putridus  which  grows  anaerobically  on  blood  agar  giving 
porcelain  white  colonies  without  haemolysin.  The  cultures  have  a  putrid  odor. 
Not  pathogenic  for  animals. 

Warren  and  Herrick  have  recently  published  a  very  important  study 
of  134  cases  of  bacteriaemia.  Bacteriaemia  signifies  the  mere  presence 
of  bacteria  in  the  blood  without  reference  to  symptoms,  while  sepsis 
denotes  conditions  due  to  invasion  of  the  blood  stream  by  bacteria  or 
their  toxins  with  marked  systemic  reaction. 

Of  25  cases  with  endocarditis  22  died  and  3  were  unimproved.  Of  55  cases  of 
sepsis  39  died  and  10  recovered.  In  postpartum  infections  10  died  and  i  recovered. 
In  osteomyelitis  5  died  and  4  recovered.  In  otitis  media  2  died  and  4  recovered. 

Thirty-one  cases  were  due  to  Streptococcus  hamolylicus  of  which  21  died. 

Forty  cases  were  due  to  S.  viridans  and  25  died.     One  case  from  S.  mucosus  died. 

Of  39  cases  with  Staphylococcus  aureus  22  died  and  in  3  cases  of  S.  albus 
2  died. 

Of  10  cases  of  Pneumococcus  bacteriemia  6  died.  Six  of  B.  coli  infection  gave  4 
deaths;  two  of  B.  influenzas  2  deaths;  three  of  anaerobic  streptococci,  2  deaths  and 
two  of  B.  mallei,  2  deaths. 

Seven  cases  of  mixed  infections  gave  6  deaths. 

The  ordinary  leukocyte  and  differential  count  procedures  were  of  very  little  value 
in  prognosis. 

The  average  white  count  in  fatal  cases  of  S.  hamolyticus  infection  was  18347 
with  81%  of  polymorphonuclears  while  in  cases  recovering  it  was  18742  and  85%, 
respectively.  Fatal  S.  viridans  infections  gave  an  average  white  count  of  15976  with 
polymorphonuclear  percentage  of  78,  while  nonfatal  cases  gave  17222  and  75%, 
respectively. 

Of  25  cases  treated  with  vaccines,  81%  died;  while  of  47  under  surgical  treatment, 
50%  died  and  with  50  treated  pallia tively,  50%  died. 

Typhoid  cultures  are  best  obtained  in  the  first  week  of  the  disease, 
after  that  time  the  Widal  is  the  test  of  preference. 

If  a  paratyphoid  serum  is  not  at  hand  for  testing,  it  may  suffice  to  inoculate  a 
glucose  bouillon  tube  or  a  Russell  lactose  glucose  litmus  slant;  gas  production 
indicates  paratyphoid.  This  test  should  be  applied  when  a  very  motile  organism 


BLOOD  CULTURES  AND  BLOOD  PARASITES         415 

does  not  show  agglutination  with  a  known  typhoid  serum.     Anthrax  and  glanders 
should  be  considered  in  blood  cultures. 

In  Malta  fever  it  must  be  remembered  that  colonies  do  not  show  themselves 
for  several  days.  Addition  of  blood  to  melted  agar  is  a  good  procedure. 

Blood  for  culturing  typhoid  or  the  paratyphoids  may  be  taken  with 
a  Wright's  tube  from  the  ear  or  finger.  Dipping  the  hand  in  hot 
water  assists  the  flow  of  blood.  The  supernatant  serum  after  centrif  u- 
galization  should  be  pipetted  off  with  a  sterile  pipette  and  reserved 
for  agglutination  tests  while  the  clot  is  dropped  into  a  bile  tube.  (Clot 
culture.) 

Rosenberger  was  the  first  to  insist  upon  the  importance  of  examination  of  blood 
for  T.  B.  Brem  considered  that  many  cases  of  finding  of  acid-fast  bacilli  were  not 
of  T.  B.  The  Kurashigi-Schnitter  method  for  tubercle  bacilli  in  blood  is  to  take 
about  i  c.c.  blood  and  put  in  a  centrifuge  tube  containing  5  c.c.  of  3%  acetic  acid. 
After  the  red  cells  are  thoroughly  laked  centrifuge,  pipette  off  supernatant  fluid  and 
dissolve  the  sediment  in  5  c.c.  antiformin.  When  dissolved  add  5  c.c.  absolute 
alcohol  and  centrifugalize  for  twenty  minutes.  Smear  out  the  sediment  and  stain. 

The  examination  of  the  blood  for  the  parasites  of  malaria,  filariases,  kala-azar 
and  spirillum  fevers  has  been  discussed  under  their  respective  headings. 

With  trypanosomes  from  human  trypanosomiasis,  smears  from  gland  juice  or 
cerebrospinal  fluid  seem  more  satisfactory  to  examine  then  blood  smears  unless  the 
blood  is  taken  in  5  to  10  c.c.  quantities  and  centrifuged  in  sodium  citrate  salt 
solution. 

The  latest  method  in  the  diagnosis  of  trichinosis  is  to  take  5  to  10  c.c.  of  blood 
from  a  vein  at  the  time  of  the  migration  of  the  embryos  to  the  muscles  (ten  to  twenty 
days).  This  is  forced  out  into  a  centrifuge  tube  containing  3%  acetic  acid,  and 
the  sediment  examined  for  trichina  larvae. 


CHAPTER  XXX 
THE  STOMACH  AND  DUODENAL  CONTENTS 

FROM  a  microscopical  standpoint  there  is  comparatively  little  that 
is  of  value  in  the  examination  of  the  gastric  contents;  there  is  nothing 
very  specific  about  the  findings. 

A  test  meal  is  not  a  necessity  as  in  the  chemical  examination,  but 
either  vomitus  or  material  withdrawn  with  a  stomach-tube  two  or  more 
hours  after  an  ordinary  meal  suffice. 

The  most  satisfactory  specimen  is  one  taken  before  the  giving  of 
the  test  meal. 

The  washings  from  the  stomach  are  allowed  to  stand  until  the 
sediment  has  fallen  to  the  bottom  and  an  examination  of  this  is  made. 

The  microscopical  diagnostic  points  in  connection  with  distinguishing  cancer  of 
the  stomach  from  nonmalignant  dilatation  are :  i .  Fragments  of  cancer  tissue.  These 
are  very  rarely  found  and  are  most  difficult  to  diagnose.  2.  The  presence  of  flagell- 
ates in  the  early  stages  of  cancer  (the  so-called  anacid  stage  preceding  the  develoy- 
ment  of  lactic  acid).  As  flagellates  prefer  an  alkaline  medium,  they  disappear 
after  the  acidity  due  to  lactic  acid  comes  on.  3.  The  presence  of  the  Boas-Oppler 
bacillus.  There  are  probably  several  organisms  so  designated.  They  are  lactic  acid 
producers  and  are  characterized  by  being  very  large  bacilli  (7XI/-0  and  arranged  in 
long  chains  which  stretch  across  the  field  of  the  microscope.  They  are  Gram-posi- 
tive and  do  not  form  spores.  They  can  be  cultivated  on  media  rich  in  milk  or  blood 
and  are  aerobic.  They  should  only  be  reported  when  present  in  great  abundance  and 
in  long  chains.  Heinemann  thinks  it  probable  that  the  Boas-Oppler  bacillus,  Lepto- 
thrix  buccalis,  and  B.  bifidus  may  be  identical  with  B.  bulgaricus  (see  under  Milk). 

4.  The  absence  of  sarcinae  and  yeasts.  The  presence  of  these  sarcinae  and  fungi  in 
vomitus  is  indicative  of  a  simple  dilatation. 

In  the  diagnosis  of  cancer,  other  than  the  finding  of  tumor,  gastric 
stasis,  etc.,  much  importance  is  attached  to  the  absence  of  free  HC1. 
This  is  best  determined  by  the  Gluzinski  test.  In  the  Mayo  clinic 
total  acidity  of  gastric  contents  averaged  63  (%  free  HC1)  in  ulcer, 
while  cancer  averaged  only  31  (%  free  HC1).  Lactic  acid  was  found 
in  43%  and  blood,  occult  or  otherwise,  in  73%  of  cancer  cases. 

In  determining  the  pepsin  activity  of  the  gastric  juice  we  usually 
employ  the  Mett  tube. 

416 


THE  STOMACH  AND  DUODENAL  CONTENTS  417 

In  chronic  gastritis  the  picture  of  mucus  entangling  large  numbers  of  cells  is 
characteristic.  It  must  be  remembered,  however,  that  strands  of  mucus  entangling 
polymorphonuclear  cells  may  be  found  in  normal  gastric  contents.  The  pus  cells 
which  are  free  are  the  ones  of  significance.  An  occasional  cylindrical  gastric  epithe- 
lial cell  may  be  found.  The  recognition  of  carcinoma  cells,  especially  those  showing 
mitoses,  is  of  value  only  when  done  by  one  who  has  made  a  special  study  of  the 
findings. 

In  examining  the  sediment  from  the  filter-paper  after  filtering  off  the  stomach 
contents  always  use  a  dilute  Gram  solution  (about  i  to  4)  for  mounting  the  sediment. 
Muscle  fibers,  yeast  cells,  red  blood-cells,  and  epithelial  cells  are  stained  a  golden 
yellow.  Starch  granules  are  stained  blue  while  fats  are  unstained  and  show  as 
globules  of  varying  sizes. 

The  Duodenal  Fluid.— This  is  best  obtained  with  the  Jutte  modi- 
fication of  the  Einhorn  duodenal  tube.  There  is  little  difficulty 
in  the  passage  of  the  tube  when  the  stomach  is  not  dilated.  One  can 
aspirate  from  5  to  20  c.c.  in  about  five  minutes,  the  amount  varying  with 
the  individual  case.  The  normal  fluid  is  clear,  bile  stained,  sero- 
mucous  and  contains  few  cells  and  living  bacteria.  Dead  bacteria  may 
be  found  in  considerable  numbers. 

Of  the  greatest  importance  is  the  chemical  examination  of  duodenal  fluid  for 
pancreatic  ferments.  It  is  well  recognized  that  the  examination  of  the  faeces  for 
such  ferments  is  absolutely  unreliable  while  with  duodenal  fluid  it  is  most  satisfac- 
tory. The  normal  ferment  value  of  duodenal  fluid  is  best  indicated  by  the  tryptic 
power,  this  being  very  constant.  The  lipase  findings  are  very  variable. 

Normally  10  c.c.  of  0.1%  casein  solution  is  digested  in  twenty-four  hours  by 
duodenal  fluid  in  dilutions  of  i  to  3000  to  i  to  10,000  or  even  higher. 

Turbidity  of  the  fluid  is  thought  to  be  suggestive  of  cholelithiasis. 
Bile-stained  pus  cells  are  also  of  significance  in  cholecystitis.  Hence 
examine  sediment  unstained  before  adding  Gram's  iodine  solution. 
The  presence  of  bile  in  the  duodenal  fluid  is  a  far  more  certain  index 
of  the  patency  of  the  bile  duct  than  the  test  of  faeces  for  bile  as  bile 
may  be  absent  in  a  fasces  test  when  present  in  duodenal  fluid.  The 
color  of  the  fluid  is  about  as  satisfactory  an  indication  of  the  presence 
of  bile  as  that  given  by  the  various  tests  for  bile.  Einhorn  states  that 
with  turbid  bile  cholecystitis  with  gall-stones  is  almost  always  present 
if  with  fasting  condition.  Gall-stones  without  cholecystitis  may  give 
a  clear  fluid  Liver  conditions,  as  neoplasms  or  high  grade  cirrhosis 
may  give  rise  to  turbid  duodenal  fluid. 

McNeil  attaches  importance  to  the  Wolff- Junghan's  test  of  the  duodenal  fluid 
which  he  states  gives  turbidity  up  to  the  third  or  fourth  tube,  but  never  in  the  fifth 
or  sixth   one,  normally.     An  increase  would  indicate  some  pancreatic,  biliary  or 
duodenal  inflammation,  provided  we  checked  up  the  test  on  the  gastric  contents. 
27 


4i8 


DUODENAL  FLUID 


In  duodenitis  we  have  stringy  mucus  and  rather  considerable  numbers  of  livii 
bacteria. 

Typhoid  carriers  are  best  recognized  by  culturing  the  duodenal  contents.  Mac- 
Neal,  in  culturing  the  organisms  from  the  duodenal  contents  of  26  cases,  found  that 
few  living  bacteria  were  present  although  dead  ones  might  be  present  in  considerable 
numbers.  The  living  organisms  are  as  a  rule  Gram-positive  cocci. 

B.  lactis  aero  genes  was  found  rather  constantly  by  Gessner. 

In  connection  with  the  possible  significance  of  bacteria  in  the  duo- 
denal contents  it  is  interesting  to  note  the  findings  of  Kelly  in  a  bac- 
teriological study  of  413  cases  of  operations  on  the  biliary  tract  by 
Deaver.  About  one-half  the  cases  showed  sterile  bile  but  in  the  acute 
cases  living  bacteria  were  almost  constantly  present.  B.  coli  was 
found  in  30%,  B.  typhosus  in  7%,  Staphylo coccus  pyogenes  in  2.9%, 
Streptococcus  in  0.2%.  Other  organisms  were  more  rarely  found  and 
55%  were  sterile. 

In  recently  studying  the  bacteria  of  the  duodenal  contents  and  subsequently  t 
bile  of  a  case  of  cholelithiasis  with  cholecystitis  the  only  organism  found  was  B.  coli. 
This  organism  was  markedly  haemolytic  on  blood  agar  plates. 

For  the  carrying  out  of  the  various  tests  see  appendix. 


CHAPTER  XXXI 
EXAMINATION  OF  PUS 

Pus  may  be  collected  for  examination  either  i.  with  a  platinum  loop, 
2.  with  a  sterile  swab,  3.  with  a  bacteriological  pipette  or  4.  with  a 
hypodermic  syringe. 

It  is  always  well  to  make  a  smear  and  stain  it  by  Gram's  method  at 
the  same  time  that  cultures  are  made.  The  Gram  stain  gives  informa- 
tion as  to  the  abundance  of  organisms  in  the  pus  and  as  to  the  probable 
findings  in  the  culture.  Pneumococci  and  streptococci  are  differenti- 
ated from  the  staphylococci  in  this  way  without  the  necessity  of  more  or 
less  extended  cultural  methods. 

Smears  from  material  examined  for  gonococci  may  show  Gram-negative  diplo- 
cocci  which,  however,  do  not  generally  have  the  morphology  of  the  Gonococcus. 
They  are  furthermore  extracellular. 

The  M.  catarrhalis  has  been  reported  from  urethral  smears  though  very  rarely. 
Diphtheroid  organisms  are  not  uncommon.  Gram-positive  cocci  are  rather  common 
in  smears  from  discharges  of  chronic  gonorrhoea. 

When  autogenous  vaccines  are  to  be  made,  the  isolation  of  the  exciting  organism 
is  necessary.  This  is  best  done  by  streaking  the  pus,  taken  up  with  a  sterile  swab 
and  emulsified  in  a  tube  of  bouillon,  over  the  surface  of  an  agar  plate.  Practically 
as  convenient  and  providing  a  more  nutritious  medium  is  to  smear  the  material  on 
a  loop  or  swab  over  the  surface  of  a  blood-serum  slant,  then  to  inoculate  a  second 
tube  from  the  first  without  recharging  the  loop  or  swab,  and  so  on  until  three  or  four 
tubes  are  inoculated.  Isolated  colonies  should  be  obtained  in  a  third  or  fourth  tube. 

In  examining  blood-serum  slants  inoculated  with  purulent  material,  always 
examine  the  water  of  condensation  for  streptococci. 

A  bacteriological  pipette  is  very  useful  when  pus  is  to  be  sent  to  a  laboratory; 
the  tip  can  be  sealed  in  a  flame  and  the  cotton  plug  at  the  other  end  insures  the 
noncontamination  of  the  contents.  The  material  may  be  drawn  up  either  with  the 
mouth  or  with  a  rubber  bulb. 

The  hypodermic  syringe  is  very  useful  in  puncturing  buboes,  etc., 
especially  in  plague.  A  small  pledget  of  cotton  on  a  toothpick  dipped 
into  pure  carbolic  acid  and  touched  to  a  spot  over  the  bubo,  which  after 
about  thirty  seconds  is  soaked  with  alcohol,  makes  a  sterile  anaesthetic 
spot  at  which  to  introduce  the  needle  of  the  syringe.  It  must  be  remem- 
bered that  when  plague  buboes  begin  to  soften,  the  plague  bacilli  may 

419 


420 


BUCCAL  AMCEB.E 


be  replaced  by  ordinary  pus  organisms.  The  pus  from  wounds  in- 
fected with  anaerobes  is  usually  very  foul.  The  most  important  anae- 
robe in  the  discharge  from  gas  gangrene  wounds  is  B.  perfringens. 
The  pus  from  the  necrotic  center  of  climatic  bubo  is  sterile. 

It  is  remarkable  how  frequently  we  get  pure  cultures  from  abscess  material.  In 
purulent  material  from  abdominal  abscesses  we  are  apt  to  obtain  mixed  cultures, 
especially  the  colon  bacillus  and  B.  pyocyaneus,  in  addition  to  ordinary  pus  organisms. 

When  it  is  a  question  between  streptococci  and  pneumococci,  it  is  well  to  inocu- 
late a  mouse;  the  capsulated  pneumococci  at  the  autopsy  make  the  diagnosis. 

Animal  inoculation  is  also  necessary  in  plague  and  glanders,  and  possibly  anthrax. 
When  tetanus  is  suspected,  it  should  be  examined  for  as  described  under  Tetanus. 
Tuberculosis  should  also  be  identified  by  inoculating  a  guinea-pig,  as  well  as  by  acid- 
fast  staining  and  culture,  if  there  is  any  doubt  as  to  the  nature  of  the  material. 

The  black  or  yellow  granules  of  madura  foot,  as  well  as  those  of  actinomycosis, 
should  be  examined  as  recommended  in  the  section  on  fungi. 

Amoebae,  coccidia,  and  larval  echinococci  may  be  found  in  purulent  material, 
as  may  also  various  other  animal  parasites,  as  fly  larvae,  sarcopsyllae,  etc. 

The  pus  from  an  amoebic  abscess  of  the  liver  is  as  a  rule  sterile  when  cultured. 

The  examination  at  the  time  of  operation  or  exploration  frequently 
shows  an  absence  of  amoebae  as  well  as  of  bacteria.  Two  or  three  days 
later  amoebae  may  be  found  in  the  pus  draining  from  the  abscess  cavity. 

Flukes,  round  worms,  and  whip  worms  may  as  a  result  of  their  wandering  from 
the  intestinal  lumen  cause  abscesses. 

Serious  ulcerations  may  follow  infection  with  the  Guinea-worm. 
Abscesses  of  filarial  origin  are  to  be  thought  of. 


CHAPTER  XXXII 
SKIN  INFECTIONS 

CULTURAL  methods  are  as  a  rule  to  be  preferred  in  the  bacterio- 
logical examination  of  the  skin. 

This  is  best  done  by  washing  the  surface  to  be  examined  with  soap  and  water, 
in  order  to  eliminate  chance  organisms  which  may  have  settled  on  the  surface  of 
the  skin  in  dust  or  as  a  result  of  contact  with  material  containing  them.  Scrapings 
are  then  made  with  a  sterile  dull  scalpel,  and  this  material  is  emulsified  in  a  drop  of 
sterile  water  in  the  center  of  a  Petri  dish.  A  tube  of  melted  agar  at  42°C.  is  then 
poured  on  the  inoculated  drop  and,  by  mixing,  the  bacterial  flora  is  distributed  over 
the  entire  surface  of  the  plate.  Of  the  colonies  developing  on  such  plates  probably 
80%  will  be  found  to  be  staphylococci,  and  of  these  the  greater  proportion  will 
be  staphylococci  showing  white  colonies. 

Occasionally  the  aureus  or  citreus  may  be  isolated. 

Streptococci  and  colon  bacilli  are  rarely  found. 

The  Staphylococcus  pyogenes  aureus  is  the  organism  usually  isolated  from  furuncles, 
circumscribed  abscesses,  and  carbuncles. 

Streptococci  are  the  organisms  to  be  expected  in  phlegmonous  infections. 

Cold  abscesses,  which  are  frequently  due  to  tuberculous  infection,  are,  as  a  rule, 
sterile. 

Acne  pustules  may  show  staphylococci  or  the  microbacillus  of  acne  may  be  present. 

The  Bacillus  acnes  is  a  short  broad  bacillus  often  showing  a  beaded 
appearance  when  stained  by  Gram's  method.  It  is  Gram-positive. 
According  to  Hartwell  it  grows  readily  on  glucose  agar  when  cultivated 
anaerobically  (Wright's  method).  Colonies  appear  in  four  to  five  days. 

Sabouraud's  medium  for  its  culture  is:  Peptone  20  grams,  glycerine  20  grams, 
glacial  acetic  acid  5  drops,  agar  15  grams  and  water  1000  c.c.  The  bottle  bacillus, 
which  morphologically  resembles  a  yeast,  is  considered  to  be  the  cause  of  dry  pityri- 
asis  capitis.  It  may  also  be  found  in  the  comedones  of  children. 

In  the  tropics,  an  organism  which  at  times  produces  lesions  similiar  to  impetigo 
and  again  pemphigoid  eruptions  and  at  other  times  widespreading  erysipelatous 
conditions  gives  cultural  characteristics  similar  to  S.  pyogenes  aureus.  It  is  probably 
only  a  virulent  aureus.  It  has  been  described  under  the  name  of  Diplococcus  pem- 
phigi  contagiosi. 

The  Staphylococcus  epidermidis  albus,  or  stitch  abscess  coccus,  is  considered  by 
Sabouraud  to  be  the  cause  of  eczema  seborrhoicum. 

It  is  in  scrapings  from  the  skin  of  lepromata  that  we  find  acid-fast 
organisms  in  the  greatest  profusion.  In  tuberculosis  of  the  skin  the 

42  T 


422 


THE   SKIN 


tubercle  bacilli  are  exceedingly  scarce.  Inoculation  of  a  guinea-pig 
will  probably  give  positive  results  with  the  tubercle  bacillus.  The 
leprosy  bacillus  is  noninoculable  for  experimental  animals. 

Anthrax  and  glanders  cause  skin  lesions  which  can  only  be  surely  diagnosed 
culturally  or  by  animal  inoculation. 

Plague  bacilli  may  be  isolated  from  the  primary  vesicles  appearing  at  the  site  of 
the  flea  bite. 

Tropical  phagedaena  is  thought  by  some  to  be  due  to  a  sort  of  diphtheroid  or- 
ganism. The  organisms  of  Vincent's  angina  may  cause  tropical  ulcer.  Herpes 
zoster  has  been  reported  by  Rosenow  as  most  probably  due  to  a  streptococcus  with 
special  affinity  for  the  ganglia  and  posterior  roots. 

The  skin  diseases  due  to  fungi  are  discussed  under  that  section.  Of  the  skin 
affections  caused  by  animal  parasites,  ground  itch  is  the  most  important.  This  is  a 
form  of  dermatitis  due  to  the  irritation  set  up  by  the  hook-worm  larvae  penetrating 
the  skin  of  the  foot  and  leg. 

The  Sarcopsylla  penetrans  or  jigger  (sand  flea)  is  an  important  agent  in  ulcera- 
tions  about  the  foot. 

Certain  acarines  cause  skin  lesions,  as  is  also  the  case  with  the  larvae  of  certain 
flies. 

'fhe  itch  mite  (Sarcoptes  scabiei)  is  an  important  animal  parasite  of  the  skin. 

The  various  lice,  fleas  and  bedbugs  are  well  understood  as  causes  of  skin  irrita- 
tion. 

Filarial  infections  are  also  important  especially  the  ulcers  of  the 
Guinea-worm,  Calabar  swellings  of  F.  loa,  the  cystic  tumors  of  F. 
volvulus  and  the  varicose  groin  glands  and  elephantiasis  of  F.  bancrofti. 

Leeches,  as  H.  ceylonica,  may  cause  serious  ulceration. 

Oxyuris  may  cause  a  severe  irritation  about  the  region  of  the  groin  and  inner 
surfaces  of  the  thigh,  and  especially  about  the  vulvar  region  of  female  children. 

Gnathostomum  siamense,  a  nematode  with  two  lip-like  structures  and  spine- 
like  appendages  covering  its  anterior  one-third,  has  been  found  once  in  a  tumefac- 
tion of  the  breast. 

Plerocercoid  larvae  of  Dibothriocephalidae  have  been  found  in  the  subcutaneous 
tissues. 

Certain  skin  diseases,  as  Oriental  sore  and  yaws,  are  protozoal  in  origin.  The 
cutaneous  lesions  of  uta  or  espundia  are  now  known  to  be  caused  by  a  Leishmania 
as  well  as  Oriental  sore.  These  affections  in  the  Central  and  South  American 
countries  are  now  known  as  American  leishmaniases. 

Dew  itch  or  foot  itch  is  caused  by  the  penetration  into  the  skin  about  the  toes  of 
the  strongyloid  encysted  larva  of  the  hook-worm. 


CHAPTER  XXXIII 
CYTODIAGNOSIS  AND  SPINAL  FLUID  EXAMINATIONS 

THIS  method  of  diagnosis  is  chiefly  employed  in  the  examination  of 
cellular  sediments  of  pleural,  ascitic,  and  cerebrospinal  fluid. 

The  fluids  which  pathologically  collect  in  the  serous  cavities  are 
divided  into  two  classes,  i.  the  transudates,  which  form  as  the  result 
of  some  circulatory  inadequacy  and  2.  the  exudates,  which  result  from 
inflammatory  processes. 

Transudates  have  little  or  no  fibrin  and  very  few  cellular  elements  and  do  not 
contain  nucleo-albumin.  Exudates  contain  nucleo-albumin  and  usually  have  a 
specific  gravity  above  1018,  while  that  of  the  transudates  is  lower  than  1018. 

There  are  two  simple  methods  for  differentiating  transudates  and  exudates. 
Moritz  adds  2  drops  of  a  5%  solution  of  acetic  acid  to  the  fluid  to  be  tested.  A 
heavy,  cloud-like  precipitate  shows  the  fluid  to  be  of  inflammatory  origin  (an 
exudate).  A  transudate  may  produce  a  slight  opalescence.  Rivalta's  test  consists 
in  dropping  a  drop  of  the  fluid  to  be  tested  into  a  cylinder  containing  2  drops  of 
glacial  acetic  acid  in  100  c.c.  distilled  water.  A  nebulous  cloud  as  the  drop  of  fluid 
sinks  shows  an  exudate. 

For  pleural  fluids  we  should  receive  the  material  in  centrifuge  tubes  about  one- 
fourth  filled  with  2%  sodium  citrate  salt  solution.  This  prevents  clotting.  Having 
thrown  down  the  sediment,  the  supernatant  fluid  is  poured  off,  and  in  its  place  a  i% 
aqueous  solution  of  formalin  is  added.  After  mixing  and  allowing  to  stand  for  about 
five  minutes,  centrifugalization  is  again  repeated  and,  pouring  off  the  supernatant 
formalin  solution,  we  make  smears  from  the  sediment.  This  is  either  stained  by  a 
Romano wsky  method  or,  after  fixing  with  heat  (burning  alcohol),  the  smear  is 
stained  with  haematoxylin  and  eosin. 

With  ascitic  fluid  it  is  usually  sufficient  to  centrifuge  the  fluid,  then  decant  off  the 
supernatant  fluid  and  drain  by  means  of  a  piece  of  filter-paper  held  at  the  mouth 
of  the  upturned  tube.  The  sediment  adheres  to  the  bottom  of  the  tube  and  is 
best  emulsified  with  the  small  amount  of  fluid  remaining  by  means  of  a  bulb  pipette. 
The  material  is  sucked  up,  smeared  out  on  a  slide  with  a  second  slide  as  for  blood 
and  stained  preferably  with  Giemsa  after  fixation.  H.  E.  staining  brings  out 
mitotic  figures  best.  If  the  fluid  has  coagulated  it  is  best  to  take  a  little  of  the 
coagulum  and  stain  it  with  neutral  red  as  for  vital  staining.  It  is  difficult  to  disso- 
ciate the  cells  from  the  clot.  I  now  make  it  a  rule  to  collect  a  portion  of  the  pleural 
or  ascitic  fluid  in  citrated  salt  solution  in  order  to  prevent  coagulation.  The  mate- 
rial is  then  centrifuged  and  after  removal  of  supernatant  fluid  with  a  bulb  pipette 
the  cell  sediment  is  drawn  up  and  smeared  out  on  a  slide  for  cell  or  bacterial  staining. 

423 


424  CYTODIAGNOSIS 

By  making  smears  as  for  blood  beautiful  preparations  may  be  obtained.  I  prefer 
Giemsa  for  differentiating  cells  and  Gram's  staining  for  bacteria. 

The  wet  Giemsa  method  described  for  blood  gives  good  results  with  puncture 
fluid  sediments. 

At  the  time  of  securing  fluid  for  cytodiagnosis,  cultures  should  be  made  on  blood- 
serum  for  various  pyogenic  bacteria  and,  if  tuberculosis  is  suspected,  inoculation 
of  a  guinea-pig  is  indicated. 

The  interpretation  of  cellular  sediments  is  more  difficult  than  many 
books  would  indicate,  there  being  many  factors  which  tend  to  compli- 
cate the  findings. 

The  polymorphonuclears  in  purulent  fluids  often  show  fatty  degeneration,  swollen 
and  faintly  staining  nucleus  or  a  breaking  up  of  the  nucleus  into  small  deeply 
staining  masses  (nuclear  fragmentation).  Such  fragments  in  the  smear  may  be  con- 
fusing. The  endothelial  cells  often  show  fatty  degeneration  in  the  cytoplasm  and 
we  often  note  bacteria  and  other  cells  which  have  been  phagocytized  by  them. 
Where  proliferation  of  endothelial  cells  is  going  on  actively  the  cells  show  a  rather 
deeply  staining  cytoplasm  as  compared  with  the  light  staining  cytoplasm  of  the  cells 
in  transudates.  Some  authorities  attach  importance  to  the  Foulis'  cells  in  connec- 
tion with  malignant  processes  in  the  peritoneum;  often  those  associated  with  malig- 
nant types  of  ovarian  cysts.  Such  cells  are  large,  often  multinucleated  and  may 
show  appearance  as  if  budding. 

The  following  are  the  leading  differentiations: 

1.  A  smear  showing  almost  entirely  lymphocytes  with  a  few  red 
cells  and  very  rarely  a  polymorphonuclear  indicates  a  tuberculous 
process. 

2.  Where  a  pyogenic  process  is  engrafted  on  a  tuberculous  one,  we 
have  still  the  red  cells,  some  degenerated  lymphocytes,  and  in  particular 
polymorphonuclears  showing  fragmentation  of  their  nuclei. 

3.  When  a  hydrothorax  results  from  chronic  hear  tor  kidney  disease, 
the  characteristic  cell  is  the  endothelial  cell,  which  greatly  resembles  a 
large  mononuclear.     These  cells  often  are  arranged  in  plaques. 

4.  Some  authorities  consider  that  the  cancer  cell  can  be  recognized 
by  its  occurring  in  masses  and  having  a  markedly  vacuolated  cytoplasm. 
It  has  been  claimed  that  they  contain  glycogen  by  which  means  we  can 
distinguish  them  from  endothelial  cells  which  they  so  much  resemble. 
If  such  cells  should  show  mitosis  the  finding  would  be  suggestive. 
For  mitotic  figures  wet  fixation  with  some  bichloride  fixative,  with  H.  E. 
staining,  is  best. 

Jousset  introduced  inoscopy  as  a  means  of  diagnosing  tuberculosis.  The  fluid 
was  allowed  to  coagulate  and  was  then  digested  with  an  artificial  gastric  juice.  The 
digested  material  was  then  centrifuged  and  the  sediment  examined  for  tubercle 


CYTODIAGNOSIS  AND  SPINAL  FLUID  EXAMINATIONS  425 

bacilli.    This  process  does  not  seem  to  have  met  with  much  favor  in  this  country. 
(Using  sodium  citrate  obviates  the  necessity  for  digesting  the  coagulum.) 
The  same  points  will  hold  for  ascitic  fluid  as  for  pleural  fluid. 

CEREBROSPINAL  FLUID  EXAMINATIONS 

In  taking  cerebrospinal  fluid  for  culture  and  cytodiagnosis  we  use  a 
stout  antitoxin  needle  without  attaching  a  syringe.  Aspiration  is 
responsible  for  many  of  the  ill  effects  of  lumbar  puncture. 

The  needle  should  be  about  4  inches  long  for  an  adult.  Sterilize  the  skin  and  needle 
as  described  for  blood  cultures  from  a  vein.  To  make  a  lumbar  puncture,  place 
patient  on  left  side  with  knees  drawn  up.  A  line  at  the  level  of  the  iliac  crests  passes 
between  the  third  and  fourth  lumbar  vertebrae.  Select  a  point  midway  between  the 
spinous  processes  of  these  lumbar  vertebrae  and  enter  the  needle  two-fifths  of  an 
inch  to  the  right  of  this  point,  pushing  the  needle  inward  and  upward.  Collect 
the  material  in  a  sterile  test-tube.  Make  cultures  on  blood-serum  and  then  cen- 
trifugalize  and  examine  the  sediment  as  for  pleural  fluids. 

Cell  Count. — A  method  of  examination  considered  by  neurologists 
as  of  differential  diagnostic  value  is  to  count  the  number  of  cells  in  a 
cubic  millimeter  of  the  cerebrospinal  fluid.  The  technic  is  to  use  a 
gentian- violet- tinged  3%  solution  of  acetic  acid.  This  is  drawn  up  to 
the  mark  0.5,  and  the  cerebrospinal  fluid  is  then  sucked  up  to  n. 
After  mixing,  the  cell  count  is  made  with  the  haemocytometer.  Nor- 
mally we  have  only  one  or  two  cells  per  cubic  millimenter,  but  in  tabes 
or  general  paresis  this  is  increased  to  50  or  100  cells  (greatest  at  onset  of 
disease). 

It  is  advisable  to  make  the  cell  count  of  the  fluid  as  soon  after  obtaining  it  as 
possible,  the  cells  tending  to  degenerate.  It  is  customary  to  consider  fluid  contain- 
ing blood  as  unsatisfactory  for  the  cell  count  as  well  as  for  the  globulin  tests,  but  one 
can  calculate  the  leukocytes  due  to  blood  content  by  counting  the  red  cells  and  sub- 
tracting one  leukocyte  for  each  750  red  cells. 

It  is  now  generally  recommended  to  make  the  spinal  puncture  with  the  patient 
seated  on  a  stool  with  the  shoulders  inclined  forward  thus  giving  the  greatest  space 
between  the  spinous  processes.  After  the  punpture  the  patient  should  drink  a 
glass  or  so  of  water  and  remain  in  bed  for  a  day.  In  some  clinics  the  subjects  lie 
down  for  a  few  hours  and  then  return  to  their  homes. 

In  general  terms  it  may  be  stated  that: 

1.  A  lymphocytosis  indicates  a  tuberculous  or  poliomyelitis  process. 

2.  An  abundance  of  polymorphonuclear  and  eosinophilic  leukocytes 
indicates  a  meningococcic,  streptococcic,  influenza  or  pneumococcic 
infection.     The  fluid  in  (i)  tends  to  be  clear,  that  in  (2)  cloudy. 


426 


LANGE'S  PARESIS  TEST 


When  the  case  is  one  of  meningism  there  are  very  few  cells.  In  poliomyelitis 
there  is  a  cell  increase  of  which  90%  may  be  lymphocytes. 

Trypanosomiasis  gives  a  cellular  increase  very  similar  to  syphilis. 

In  the  work  of  the  French  Sleeping  Sickness  Commission  five  cells  per  cubic 
millimeter  was  taken  as  normal. 

Miller  gives  the  following  table  as  to  pleocytosis: 

AVERAGE  INCIDENCE  OF  LYMPHOCYTOSIS  IN  THE  SPINAL  FLUID 
(Plaut,  Rehm  and  Schottmuller) 


CLINICAL  DIAGNOSIS 
Cerebrospinal  lues  

FREQUENCY 
85-90% 

REMARKS 

Counts  often  over  100  —  may 
c.mm. 

- 

reach  1000  per 

Tabes  dorsalis 

00% 

Counts  usually  under  100. 

General  paresis 

08% 

Counts  average  30—60  cells  pe 

r  c.mm. 

Secondary  lues 

7Q—  AO% 

Moderate  increase  as  a  rule. 

Multiple  sclerosis  

25% 

Border-line  counts. 

Cerebral  haemorrhage  . 
Cerebral  tumors  
Sinus  thrombosis  

f  Frequency 
is 
[   variable 

Cellular  increase  is  apt  to  be  a 
one. 

very  moderate 

Colloidal  Gold  Test  (Lange's). — It  is  now  generally  accepted  that 
this  test  is  more  diagnostic  of  general  paresis  than  any  other  single 
test.  The  color  changes  in  the  first  five  tubes  (i-io;  1-160)  are  so 
constant  that  the  term  "paretic  curve"  is  applied  to  such  findings. 
Of  less  diagnostic  value  are  the  so-called  cerebrospinal  lues  curves 
where  the  color  changes,  though  of  less  intensity  than  the  paretic  ones, 
are  most  marked  in  the  third,  fourth,  fifth  and  sixth  tubes  (1-40  to  1-320). 
In  various  types  of  meningitis,  other  than  luetic,  the  color  changes  are 
at  times  more  marked  in  the  tubes  with  the  higher  dilutions  of  spinal 
fluids  (from  1-320  to  1-2560). 

The  paretic  curve  of  the  colloidal  gold  test  generally  runs  parallel  with  a  spinal 
fluid  Wassermann  and  globulin  increase.  This  agreement  does  not  exist  at  all  con- 
stantly for  positive  blood-serum  Wassermann  tests  and  increased  cell  counts. 

It  may  be  stated  that  this  test  is  of  more  importance  in  paresis  than  any  single  one 
of  the  four  reactions  of  Nonne,  viz.  (a)  blood-serum  Wassermann  (b)  spinal  fluid 
Wassermann  (c)  globulin  increase  and  (d)  increased  cell  count  of  spinal  fluid  (pleo- 
cytosis). Of  course,  all  of  these  tests  should  be  carried  out. 


CYTODIAGNOSIS  AND  SPINAL  FLUID  EXAMINATIONS  427 

Test.  Put  1 1  clean  dry  test-tubes  in  a  rack  and  deposit  in  the  first  tube  1.8  c.c. 
of  a  0.4%  solution  of  sterile  saline.  Into  the  other  10  tubes  put  only  i  c.c.  of  the 
0.4%  saline.  Into  the  first  tube  deliver  0.2  c.c.  of  spinal  fluid  and  mixing 
thoroughly  we  have  2  c.c.  of  a  i-io  dilution.  Withdraw  i  c.c.  from  the  first  tube 
and  add  to  the  i  c.c.  of  saline  in  the  second  tube.  This  gives  2  c.c.  of  1-20. 
Continue  the  process  until  the  No.  i  to  No.  10  tubes  contain  i  c.c.  quantities  of  the 
various  dilutions  from  i-io  to  1-5120. 

Tube  ii  contains  no  spinal  fluid  but  only  i  c.c.  of  the  saline  and  serves  as  a 
control. 

To  each  of  these  n  tubes  add  5  c.c.  of  the  colloidal  gold  reagent.  The  color 
changes  are  usually  read  after  the  tubes  have  stood  over  night  at  room  tem- 
perature. The  proper  color  of  the  control  in  tube  n  should  be  salmon  red  or  old 
rose  and  the  fluid  should  be  perfectly  transparent.  When  the  color  is  changed  in 
tubes  containing  dilutions  of  the  spinal  fluid  we  record  one  showing  a  bluish  tint 
as  i.  When  the  change  is  to  a  lilac  we  record  it  as  2.  A  distinct  blue  is  marked 
as  3  and  a  pale  blue  as  4.  When  decolorization  is  complete  there  is  the  highest 
color  change,  which  is  noted  as  5. 

All  glass-ware  used  in  the  test  should  be  thoroughly  washed  in  soap  and  hot  water 
and  then  carefully  rinsed  with  tap  water.  Next  use  the  bichromate-sulphuric  acid 
cleansing  fluid,  followed  by  most  thorough  washing  in  running  water  followed  by 
distilled  water. 

In  preparing  the  reagent  a  2  liter  glass  beaker,  following  the  above-described 
cleansing,  is  rinsed  in  double  or  triple  distilled  water,  made  with  block  tin  condens- 
ing tubes  and  without  rubber  connections.  Then  fill  the  beaker  with  triple  dis- 
tilled water  up  to  a  ^  liter  mark.  Heat  the  water  gradually  to  6o°C.  Now 
add  5  c.c.  of  a  i%  aqueous  solution  of  Merck's  yellow  crystalline  gold  chloride 
and  3^  c.c.  of  a  2%  aqueous  solution  of  desiccated  potassium  carbonate.  Con- 
tinue the  heating  of  the  solution,  which  should  remain  clear,  to  8o°C.,  then 
add  5  drops  of  a  i%  aqueous  solution  of  oxalic  acid,  stirring  all  the  time.  The  solu- 
tion should  be  colorless  after  adding  the  oxalic  acid.  When  the  temperature  reaches 
QO°C.  remove  the  flame  and  add  drop  by  drop  5  c.c.  of  i%  formalin  solution, 
stirring  continuously.  Should  a  pink  color  show  itself  before  all  the  formalin  solu- 
tion has  been  added  stop  the  further  addition.  It  soon  assumes  the  required  shade 
and  when  cool  should  be  perfectly  transparent  and  of  an  old  rose  or  orange-red  color. 

Globulin  Increase  Tests. — The  test  generally  used  is  Noguchi's  buty- 
ric acid  one.  Deliver  into  a  small  test-tube  0.5  c.c.  of  a  10%  solution 
of  butyric  acid  in  0.9%  salt  solution.  Then  add  o.i  c.c.  of  spinal  fluid. 
Bring  to  a  boil  over  a  flame  and  add  o.i  c.c.  of  N/i  NaOH  solution. 
If  there  is  a  considerable  increase  of  globulin  a  flocculent  precipitate 
appears  in  a  few  minutes  or  at  any  rate  in  one  or  two  hours.  Fluids 
with  a  normal  content  or  only  slight  increase  only  show  a  slight  opacity. 

The  odor  of  the  butyric  acid  is  very  objectionable  and  in  our  laboratory  we  use 
the  Ross-Jones  method.  In  this  one  deposits  in  a  small  tube  about  i  c.c.  of  satu- 
rated solution  of  ammonium  sulphate.  On  the  surface  of  this  column  we  deposit 
i  c.c.  of  spinal  fluid.  If  globulin  increase  is  present  a  turbid  ring  appears  within 


428 


GLOBULIN  INCREASE  TESTS 


"• 


a  few  seconds  at  the  junction.     Normally  there  is  no  sign  of  a  ring.     This  test 
modification  of  Nonne's  Phase  I  reaction. 

A  test  that  is  not  in  general  use  is  strongly  recommended  by  Miller.  It  is  known 
as  Pandy's  test.  To  carry  it  out  prepare  a  saturated  solution  of  carbolic  acid  crystals 
in  distilled  water.  Place  i  c.c.  of  this  reagent  in  a  small  test-tube  and  add  i  drop 
of  spinal  fluid.  In  a  normal  fluid  only  the  faintest  opalescence  is  observed,  but  in 
a  fluid  with  globulin  increase  a  smoke  like  white  cloud  developes  instantly  where 
the  drop  comes  in  contact  with  the  reagent.  Miller  gives  the  following  table  as 
showing  the  average  frequency  of  the  various  reactions  in  syphilis  of  the  central 
nervous  system. 

SHOWING  THE  AVERAGE  FREQUENCY  OF  THE  VARIOUS  REACTIONS  IN  SYPHILIS 
THE  CENTRAL  NERVOUS  SYSTEM 


Paresis 

Tabes   dorsalis 

Cerebrospinal 
syphilis 

Blood  Wassermann  
Spinal   fluid  Wassermann 

98-100% 
07% 

70% 
60-80% 

70-80% 
8^—00% 

Pleocytosis  

98% 

8q-oo% 

8c-Qo% 

Positive  globulin   test. 

IOO% 

QO~  (K% 

OO—  Q$% 

Colloidal  gold   test  

98-100% 

8c-oo% 

7<;-8o% 

Pare  tic 
curves 

Luetic  type 
of  curve 

Luetic 
curve 

Poliomyelitis  in  addition  to  small  mononuclear  increase  together  with  rather 
characteristic  large  mononuclear  cells  shows  a  globulin  increase. 

Urea  Content. — Any  excess  of  urea  in  the  cerebrospinal  fluid  is  a  sure 
sign  of  renal  inadequacy. 

Normally  the  urea  content  of  cerebrospinal  fluid  is  only  0.006%, 
according  to  Canti.  Cases  of  true  uremia,  and  not  renal  disease 
associated  with  cardio- vascular  disease,  show  from  o.i  to  0.6%. 
Cases  showing  over  0.3%  rarely  recover.  The  estimate  may  be  made 
from  5  c.c.  of  spinal  fluid  by  the  urease  method. 


CHAPTER  XXXIV 

RABIES,  SMALLPOX,  VACCINIA  AND  THE  FILTERABLE 

VIRUSES 

RABIES  is  a  disease  of  dogs  and  wolves,  but  is  communicable  to  man 
and  domesticated  animals.  The  virus,  whatever  it  may  be,  resides  in 
the  saliva  and  nervous  structures.  It  is  destroyed  by  a  temperature  of 
5o°C.  In  man  the  period  of  incubation  is  usually  from  three  weeks  to 
three  months,  but  may  be  shorter  or  may  extend  over  one  year. 

Bites  about  the  face  and  those  with  marked  lacerations  are  particularly  serious. 
Bites  of  rabid  wolves  give  about  four  times  as  great  a  mortality  as  those  of  dogs. 
In  the  dog  there  are  two  types  of  the  disease — dumb  rabies  and  furious  rabies. 

By  inoculating  rabbits  subdurally  with  an  emulsion  of  the  brain  or  spinal  cord 
of  a  rabid  animal,  and  successively  the  medulla  of  this  rabbit  subdurally  into  other 
rabbits,  we  finally  so  increase  the  virulence  of  the  infection  that  rabbits  die  in  six  days. 
Beyond  this  it  is  impossible  to  increase  the  virulence  and  it  is  termed  fixed  virus." 
The  pathogenic  power  of  this  virus  is  also  changed  so  that  it  is  not  apt  to  cause  rabies 
if  injected  subcutaneously.  To  attenuate  this  virus  the  spinal  cord  of  the  rabbit 
is  removed  and  is  dried  over  caustic  potash  at  a  temperature  of  23°C.  The  cord  is 
divided  into  segments  about  i  inch  in  length.  Drying  for  about  fifteen  days  seems 
to  entirely  destroy  the  virus. 

To  prepare  the  material  for  prophylactic  injections  a  small  portion  of  the  cord  is 
emulsified  with  normal  salt  solution  and  injected  subcutaneously.  The  German 
method  is  to  commence  with  a  cord  that  has  been  desiccated  only  eight  days.  At 
first  injections  are  given  daily,  and  it  is  possible  to  inject  three  days'  cords  by  the 
sixth  day.  The  immunity  is  "active"  and  the  immunizing  agent  is  a  "vaccine." 
Like  vaccine  virus  the  product  can  be  preserved  (for  probably  a  month)  by  the  use 
of  glycerine  so  that  it  is  now  possible  to  send  the  material  for  inoculation  from  the 
laboratory  preparing  it. 

The  treatment  lasts  for  about  twenty  days. 

There  are  other  methods  of  treatment  as  follows: 

i.  The  Harris  Method. — In  this  the  brain  and  cord  are  ground  up 
with  CO2  snow  and  the  frozen  tissue  dried  over  H2SO4.  The  process 
of  drying  lasts  about  two  days  and  the  virulence  of  the  virus  is  reduced 
one-half.  The  potency  of  the  virus,  when  kept  at  o°C.,  holds  for 
six  months  at  least. 

429 


430 


RABIES 


iline 


2.  The  Gumming  Method. — In  this  the  brain  is  emulsified  in  saline 
and  dialyzed.     In  this  method  the  virus  is  so  attenuated  that  injec- 
tions do  not  produce  rabies  on  intracranial  inoculation. 

3.  In  the  Hogyes  method  the  fresh  virulent  cord  is  injected  but  s 
diluted  in  strength  that  it  acts  as  does  an  attenuated  virus. 

In  the  diagnosis  of  rabies  in  dogs  it  is  preferable  to  preserve  the  animal  so  that 
the  development  of  the  symptoms  may  be  observed. 

In  case  the  dog  has  been  killed,  it  may  be  possible  to  make  a  diagno- 
sis by  means  of  the  Negri  bodies.     These  are  round  or  oval  bodies  from 


FIG.  112. — Two  nerve  cells  of  hippocampus  major  (smear  preparation)  showing 
Negri  bodies.  A,  Negri  bodies;  B,  inner  bodies  within  the  Negri  bodies.  (After 
Reichel,  American  Veterinary  Review.) 


i  to  2o/x  in  diameter,  which  may  be  found  in  the  nerve-cells,  especially 
those  of  the  cornu  ammonis  (Hippocampus  major). 

These  bodies  were  first  described  by  Negri  in  1903.  In  street  rabies  large  ame- 
boid forms  from  18  to  2$n  may  be  found,  while  in  the  nerve  tissues  of  animals  with 
"fixed"  virus  only  minute  forms,  0.5/1  or  less,  may  be  detected.  The  fact  that  the 
virus  will  pass  through  a  Berkefeld  filter  is  no  argument  against  its  protozoal 
nature.  Calkins  considers  it  to  be  of  rhizopod  affinity.  The  name  Neuroryctes 
hydrophobia  has  been  given  it.  The  bodies  are  present  four  to  seven  days  before 
the  onset  of  symptoms.  They  may  be  demonstrated  by  staining  smears  of  gray 
brain  substance  by  some  Romano wsky  method,  especially  by  the  Giemsa  stain. 
The  smears  should  be  made  by  mashing  the  thin  slice  of  gray  matter  taken  from  i. 
Cornu  ammonis,  2.  Region  of  fissure  of  Rolando — in  dog  crucial  sulcus — or  3.  Cere- 
bellum, with  a  cover-glass  against  the  slide.  Afterward  the  cover-glass  is  gently 
drawn  along  the  slide. 

The  smear  on  the  slide  is  then  fixed  in  methyl  alcohol  for  two  to  three  minutes, 


I 


RABIES,   SMALLPOX,  VACCINIA  AND  FILTERABLE  VIRUSES        431 

washed  with  water  and  covered  with  a  stain  made  by  adding  3  drops  of  Sat.  ale. 
sol.  of  basic  fuchsin  to  10  c.c.  of  distilled  water  and  then  adding  2  c.c.  of  Loffler's 
methylene-blue  solution.  The  stain  on  the  slide  is  then  steamed  gently  and  after- 
ward washed  with  water  and  dried. 

As  their  relation  to  the  nerve-cell  is  more  or  less  disturbed  by  such  a  method 
it  is  preferable  to  fix  brain  tissue  from  the  region  of  the  cornu  ammonis  for  five  to 
seven  hours  in  Zenker's  fluid,  then  to  imbed  in  paraffin  and  make  sections.  These 
are  stained  with  Giemsa's  stain  and  the  Negri  bodies  are  brought  out  as  iliac-red 
bodies  in  the  blue  cytoplasm  of  the  nerve-cells.  It  is  necessary  to  differentiate  in 
95%  alcohol. 

In  the  Lentz  method  the  3;*  sections,  after  removal  of  the  paraffin,  are  flooded 
with  absolute  alcohol.  They  are  then  stained  with  a  ^%  solution  of  eosin  in  60% 
alcohol  for  one  minute.  Wash  in  water  and  next  stain  for  one  minute  in  LofHer's 
methylene  blue.  Again  wash  in  water.  Apply  LugoFs  solution  to  the  section  for 
one  minute  and  then  differentiate  alternately  in  methyl  alcohol  and  water  until  the 
section  is  pink.  After  washing  in  water,  again  stain  with  LofBer's  blue  for  one-half 
a  minute,  then  wash  in  water  and  dry  carefully  with  filter-paper.  Now  differentiate 
in  alkaline  alcohol  (i  drop  of  a  5%  solution  NaOH  in  30  c.c.  absolute  alcohol)  until 
the  section  is  pink,  then  quickly  differentiate  in  acid  alcohol  (i  drop  50%  acetic 
acid  in  30  c.c.  absolute  alcohol)  until  a  slight  blue  outline  to  the  ganglion  cells  is 
obtained.  Treat  rapidly  with  absolute  alcohol  and  xylol  and  mount  in  balsam. 
The  Negri  bodies  show  as  light  carmine  pink  bodies  on  the  light  blue  ground  of  the 
ganglion  cells.  In  the  interior  of  the  pink  bodies  dark  blue  dots  or  rings  may  be 
observed. 

This  method  can  also  be  used  for  brain  smears. 

In  addition  to  examining  for  the  Negri  bodies,  a  rabbit  may  be  inoculated  sub- 
durally  with  a  sterile  salt-solution  emulsion  of  the  medulla  of  the  dead  dog. 

If  the  brain  and  medulla  of  the  dog  are  to  be  sent  to  a  laboratory 
for  examination  they  should  be  packed  in  ice  or  placed  in  glycerine. 
Take  of  glycerine  one  part  and  one  part  water.  Sterilize  the  diluted 
glycerine  by  boiling,  allow  to  cool,  and  drop  the  pieces  of  brain  tissue 
into  this.  This  does  not  kill  the  virus. 

When  from  advanced  putrefaction,  or  otherwise,  the  Negri  bodies  cannot  be 
found  the  changes  in  the  Gasserian  ganglia  may  give  a  diagnosis.  In  typical  lesions 
the  ganglion  cells  are  more  or  less  completely  destroyed  and  replaced  by  cells  of 
other  types. 

When  a  person  is  bitten  by  a  dog  suspected  of  being  rabid  the  following 
simple  measures  should  be  instituted.  The  dog  should  be  kept  under  observation 
in  a  safe  quiet  place  and  will  show  clinical  evidence  of  rabies  within  five  days  and 
will  die  shortly  afterward  in  case  rabies  exists.  When  the  animal  dies  the  head 
and  several  inches  of  the  neck  should  be  removed  and  packed  in  ice  and  sent  to  the 
nearest  laboratory. 

Antirabic  serum  has  been  prepared  by  injecting  sheep  with  emulsions  of  rabbits' 
cord  and  brain— at  first  intravenously,  then  subcutaneously. 


432  VACCINE  VIRUS 

The  thorough  cauterization  of  the  dog-bite  wound  with  pure  nitric 
acid,  as  soon  as  possible  after  the  bite,  is  imperative  even  when  the  Pas- 
teur treatment  can  be  given  later. 

Smallpox. — The  etiology  of  this  disease  is  very  obscure,  the  virus 
being  grouped  under  the  Chlamydozoa.  Smallpox  and  vaccinia  are 
often  classed  as  filterable  viruses.  Park,  however,  was  unable  to  pass 
the  virus  of  vaccinia  through  a  Berkefeld  filter  with  40  pounds  pressure; 
the  failure  may  have  been  due  to  lack  of  sufficient  dilution.  They  did 
find  that  the  virus  would  pass  through  the  finest  filter-paper. 

In  1892  Guarnieri  noted  cell  inclusions  in  the  cornea  of  rabbits  inoculated  with 
smallpox  and  under  the  name  Cytorrhyctes  variola,  Councilman  has  described  what 
he  regards  as  a  protozoon  invading  cell  nuclei.  Certainly  ordinary  bacteria  are  not 
concerned  in  the  etiology  of  smallpox.  The  virus  is  not  only  contained  in  the  skin 
lesions  but  also  in  nasal  and  buccal  secretions,  the  disease  being  communicable 
before  the  eruption  appears.  The  period  of  incubation  of  variola  vera  is  very  con- 
stantly twelve  days,  while  that  of  variola  inoculata  (a  method  of  prophylaxis  by 
inoculating  discharges  from  a  vesicle  or  pustule  which  preceded  the  present 
method  of  vaccination)  is  eight  days.  Monkeys  are  quite  susceptible  to  both  small- 
pox and  vaccinia  as  is  also  true  of  corneal  inoculations  of  rabbits. 

Cutaneously  the  rabbit  shows  a  typical  eruption  after  vaccination, 
but  does  not  show  characteristic  lesions  after  smallpox  inoculation. 

It  is  usually  accepted  that  vaccinia  is  simply  a  permanently  modified  smallpox 
resulting  from  animal  passage  and  it  is  stated  that  repeated  passage  of  smallpox 
virus  through  calves  produces  vaccine  virus.  Inoculation  of  calves  with  smallpox 
virus  is  a  most  uncertain  procedure  and  Park  states  that  he  has  been  unable  to  obtain 
success  after  many  such  experiments. 

Of  great  practical  importance  is  the  differentiation  of  smallpox 
and  chicken-pox.  In  efficiently  vaccinated  persons  the  cutaneous  in- 
oculation of  material  from  the  suspicious  vesicle  will  give  rise  to  a  skin 
reaction  similar  to  the  Pirquet  Tb.  one,  occurring  within  twenty-four 
hours.  If  the  vesicle  were  of  chicken-pox  no  reaction  occurs.  Heating 
the  material  to  6o°C.  for  thirty  minutes  eliminates  all  danger  and  does 
not  interfere  with  the  reaction.  (Tieche.) 

Vaccinia. — Vaccinia  is  a  disease  produced  artificially  by  the  injection 
of  vaccine  virus  obtained  from  the  calf.  The  material  for  vaccine  is 
taken  from  vesicles  about  one  week  after  the  inoculation.  The  most 
potent  material  is  in  the  pulp  at  the  base  of  the  vesicle  and  not  in  the 
lymph  which  exudes  from  the  vesicle.  The  pulp  is  ground  up  and  mixed 
with  an  equal  amount  of  glycerine,  which  acts  not  only  as  a  preservative 
but  as  a  mild  antiseptic  for  nonsporing  bacteria.  The  calves  are  autop- 


RABIES,   SMALLPOX,  VACCINIA  AND  FILTERABLE  VIRUSES         433 

sied  after  the  pulp  has  been  curetted  from  the  inoculated  skin  of  the 
abdomen  to  be  sure  that  no  disease  exists  in  the  calves.  The  virus  is 
afterward  tested  for  pus  organisms,  tetanus,  and  foot  and  mouth 
disease. 

Very  important  is  the  test  for  tetanus.  Cultures  are  grown  anaerobically  for  six 
days,  then  filtered  and  the  filtrate  inoculated  into  guinea-pigs  and  these  latter 
watched  for  ten  days  to  note  evidence  of  tetanus. 

If  found  free  from  any  harmful  germs  the  vaccine  is  then  tested  upon  children  for 
potency.  Incision  is  the  proper  method  of  vaccination,  as  by  scratching  with  a 
needle,  two  lines  i  inch  long  and  i  inch  apart.  Scarification  should  never  be 
practised. 

Guarnieri  in  1892  first  observed  small  bodies  near  the  nucleus  of  infected  epi- 
thelial cells.  He  called  them  Cytoryctes  vaccinia.  Calkins  regards  these  bodies 
as  well  as  the  Negri  bodies  as  being  rhizopods  and  the  distributed  chromatin  as 
idiochromidia  (granules  of  nuclear  chromatin  within  the  cytoplasm). 

THE  FILTERABLE  VIRUSES 

The  first  disease  of  which  the  virus  was  found  to  be  capable  of  pass- 
ing through  the  finest  porcelain  filter  was  that  of  foot  and  mouth 
disease  (Loffler  and  Frosch,  1898). 

The  filter  which  is  ordinarily  used  for  testing  for  the  passage  of  such 
disease  agents  is  the  Berkefeld  filter,  one  made  of  diatomaceous  earth. 
The  filter  should  be  new  and  sterilized  before  use.  The  material 
should  be  diluted  with  saline  before  filtering.  One  may  use  slight 
suction  from  a  filter  pump.  The  filtration  should  occupy  only  a  short 
time,  not  exceeding  two  hours. 

Of  the  infections  belonging  to  man,  in  which  such  a  passage  of  blood 
or  serum  through  the  pores  of  a  porcelain  filter,  capable  of  keeping 
back  even  such  a  small  bacterial  organism  as  that  of  Malta  fever,  but 
which  does  not  hold  back  their  virus,  we  have  the  following:  foot  and 
mouth  disease,  trachoma,  molluscum  contagiosum,  vaccinia,  variola, 
rabies,  typhus  fever,  measles,  scarlet  fever,  yellow  fever,  dengue, 
Papataci  fever,  poliomyelitis  and  coryza.  It  has  been  suggested  that 
the  virus  of  cerebrospinal  meningitis  may  be  a  filterable  one. 

Hog  Cholera  Virus. — Very  interesting  is  the  history  of  the  virus  of  hog  cholera  or 
swine  fever.  This  was  supposed  to  be  due  to  an  organism  of  the  hog  cholera  group, 
B.  aertrycke  (identical  with  B.  cholera  suis  and  B.  suipestifer).  This  organism 
belongs  to  the  "enteritidis"  group  and  is  more  common  as  a  cause  of  food  poisoning 
in  man  than  the  better  known  Gaertner  bacillus.  Recently  the  group  of  organ- 
isms, including  the  paratyphoid  B.,  but  not  A,  as  well  as  the  hog  cholera  group, 
28 


434 


FILTERABLE  VIRUSES 


has  been  designated  the  Salmonella  group.     It  is  now  known  that  the  cause  of 
most  important  fatal  disease  of  swine  is  a  filterable  virus. 

This  virus  shows  remarkable  powers  of  resistance  to  external  influences,  thus  it 
can  be  kept  for  months  in  animal  tissues.  It  is  not  destroyed  by  drying  and  with- 
stands a  temperature  of  58°C.  for  two  hours  but  not  one  of  72°C.  for  one  hour. 
Cell  inclusions  have  been  found  in  smears  from  the  conjunctivas  of  hogs  sick  with 
the  disease.  See  Chlamydozoa.  A  very  valuable  prophylactic  but  not  curative 
serum  is  found  in  the  serum  of  animals  recovering  from  the  disease  or  in  those 
immunized. 

There  are  many  other  diseases  of  this  nature  which  are  important  among  the 
domesticated  animals,  such  as  pleuropneumonia  of  cattle,  African  horse  sickness 
and  hog  cholera.  The  viruses  of  pleuropneumonia  of  cattle  and  poliomyelitis  have 
been  obtained  in  artificial  cultures.  Some  of  these  viruses  seem  related  to  bacterial 
infections  and  others  to  protozoal  ones.  These  viruses  differ  as  to  method  of  trans- 
mission, pleuropneumonia  of  cattle  being  transmitted  by  inhalation,  rabies  and 
vaccinia  by  the  cutaneous  atrium,  hog  cholera  by  ingestion  and  many  of  those  sup- 
posed to  have  protozoal  affinities,  as  yellow  fever,  Papataci  fever  and  horse  sickness 
by  mosquitoes. 

As  a  rule  these  viruses  are  destroyed  by  a  temperature  of  55°C.  in  a  few  minutes. 


CHAPTER  XXXV 

DISEASES  OF  UNKNOWN  OR  NOT  DEFINITELY  DETERMINED 

ETIOLOGY 

OF  TEMPERATE  CLIMATES 

Acute  Articular  Rheumatism. — Various  bacteria  have  been  reported 
as  cause.  The  organism  which  seems  the  most  probable  cause  is  the 
short  chain  coccus,  Micrococcus  rheumaticus  of  Triboulet  and  others. 
Inoculations  of  this  streptococcus  cause  polyarthritis  and  pericarditis. 
Poynton  and  Paine  have  cultivated  the  organism  from  the  cerebro- 
spinal  fluid  in  three  cases  where  chorea  was  present. 

The  Common  Cold. — Of  all  the  diseases  common  in  man  this  condi- 
tion has  been  surrounded  by  greater  etiological  and  epidemiological 
obscurity  than  any  other. 

We  are  inclined  to  believe  that  the  common  cold  (coryza)  sets  in  when  our  re- 
sistance is  lowered  by  alimentary  tract  disturbances,  from  exposure  to  variations 
in  temperature,  or  following  refrigeration  and  fatigue.  Of  course,  many  have  held 
that  the  common  cold  was  "catching"  but  the  evidence  offered  in  support  of  such 
a  view  has  been  academic. 

Many  bacterial  organisms  have  been  suggested  as  causative  such  as 
B.  coryza  segmentosus,  haemolytic  and  viridans  types  of  streptococci, 
M.  catarrhalis,  etc.  In  1914  Kruse  brought  forward  evidence  to  prove 
that  the  etiological  factor  in  coryza  was  a  filterable  virus.  Quite  re- 
cently Foster  has  conducted  experiments  in  which,  by  using  the  nasal 
discharge  from  typical  coryza  cases,  diluting  it  with  10  or  15  times  its 
volume  of  saline,  then  passing  through  a  small  Berkefeld  filter  and  in- 
stilling 3  to  6  drops  of  the  filtrate  into  the  nasal  cavity  of  10  well  men 
he  produced  typical  coryza  in  nine  of  these  men  in  from  eight  to  thirty 
hours.  Cultures  were  made  from  the  filtrate  following  Noguchi's 
spirocha?te  culturing  method.  The  culture  medium  surrounding  the 
piece  of  sterile  tissue  showed  turbidity  in  from  forty-eight  to  seventy- 
two  hours  and  dark-field  examination  showed  myriads  of  extremely 
active  bodies  which  were  thought  to  possess  true  motility  rather  than 
Brownian  movement. 

435 


436  POLIOMYELITIS 

Filtrates  from  these  cultures  were  instilled  into  the  nasal  cavity  of  n  men  and 
after  a  period  of  incubation  of  from  eight  to  forty-eight  hours  all  came  down 
with  coryza. 

Epidemic  Poliomyelitis. — Material  from  the  cord  of  child  with  the 
disease  when  injected  subdurally,  intravascularly,  or  into  the  peritoneal 
cavity  of  monkeys  produced  the  disease  in  the  animals  inoculated. 
The  virus  has  been  passed  through  three  generations  of  monkeys 
(Flexner). 

The  virus  has  been  found  in  the  brain,  spinal  cord,  mesenteric  and  salivary  glands 
of  monkeys  and  may  remain  in  the  nasal  mucosa  of  monkeys  as  long  as  five  months. 
This  would  indicate  the  existence  of  human  chronic  carriers.  With  the  possible 
exception  of  the  rabbit  only  man  and  the  monkey  are  susceptible.  This  would 
indicate  that  the  virus  is  directly  transferred  from  man  to  man.  The  virus  is 
highly  resistant  to  drying  and  light.  It  will  remain  alive  for  months  in  dust.  It  is 
not  sterilized  by  pure  glycerine  during  many  months  of  contact.  It  is  possibly 
transmitted  by  a  biting  fly,  Stomoxys  calcitrans.  Against  this  is  the  fact  that 
the  virus  has  not  been  found  in  the  blood. 

Flexner  and  Noguchi  have  recently  cultivated  the  virus  of  polio- 
myelitis by  employing  ascitic  fluid  to  which  had  been  added  a  fragment 
of  sterile  rabbit  kidney  and  nutrient  agar,  this  culture  medium  being 
covered  with  a  layer  of  paraffin  oil.  The  growth  is  obtained  under 
anaerobic  conditions.  The  minute  colonies  are  composed  of  globular 
or  globoid  bodies  from  0.15  to  0.3  micron  in  diameter.  These  bodies 
may  be  single  or  in  chains  or  in  masses.  In  older  cultures  bizarre  forms 
are  obtained.  Monkeys  have  been  inoculated  with  the  cultures. 

Foot  and  Mouth  Disease. — Due  to  an  ultramicroscopic  organism. 

This  is  a  highly  contagious  disease  of  cattle  characterized  by  the  appearance 
of  vesicles  in  the  mouth  and  about  the  feet  of  cattle.  Man  rarely  contracts  the 
infection  through  drinking  the  milk  of  infected  animals.  This  disease  is  of  great 
interest  as  having  been  the  first  of  the  filterable  virus  diseases  to  have  been  discovered 
(Loffler  and  Frosch  in  1898). 

Measles. — Cause  entirely  unknown.     Hektoen  has  shown  that  bl 
contains  the  virus. 

Anderson  has  found  that  the  virus  of  measles  can  pass  through  a  Berkefeld  filter 
and  loses  its  infectivity  after  heating  for  fifteen  minutes  at  55°C.  In  infecting 
monkeys  it  was  found  that  the  blood  of  patients  with  measles  was  infective  only 
just  before  and  for  about  twenty-four  hours  after  the  appearance  of  the  eruption. 
Mixed  nasal  and  buccal  secretions  were  infective  for  monkeys  for  about  forty-eight 
hours  from  the  time  of  the  eruption.  The  scales  from  desquamating  cases  were 
not  capable  of  infecting  monkeys  hence  it  was  thought  that  measles  was  no 
contagious  during  the  period  of  desquamation. 


DISEASES  OF  UNKNOWN  ETIOLOGY  437 

Mumps. — Herb  has  implicated  a  diplococcus.  Inoculations  into 
Stenson's  duct  of  monkeys  successful. 

Rabies. — Probably  the  Negri  bodies. 

Roetheln  (German  Measles). — Nothing  known. 

Scarlet  Fever. — Streptococci  seem  most  probable  cause  (S.  anginosus). 
Mallory  has  implicated  epithelial  protozoa. 

Dohle  has  reported  the  rather  constant  finding  of  basophilic  round  or  oval  inclu- 
sion bodies  in  the  polymorphonuclears.  These  findings,  which  are  brought  out  in 
stained  films,  are  present  only  during  the  first  few  days  of  the  attack  of  scarlatina. 
Other  workers  have  found  these  bodies  almost  as  constantly  present  in  diphtheria 
as  in  scarlet  fever.  They  have  also  been  found  in  other  acute  diseases  than  diph- 
theria. Klemenko  has  obtained  streptococci  from  the  blood  in  only  about  2%  of 
cases  of  scarlatina.  Quite  recently  Mallory  has  reported  diphtheroid  organisms 
as  the  cause. 

Smallpox  and  Vaccinia. — Guarnieri  and  Councilman  have  impli- 
cated epithelial  protozoa. 

Spotted  Fever  of  the  Rocky  Mountains. — Supposed  to  be  due  to  an 
unknown  protozoon  transmitted  by  a  tick,  D.  andersoni. 

This  disease  is  especially  prevalent  in  the  Bitter  Root  Valley  of  Montana  and 
to  some  extent  in  the  mountains  of  Idaho.  It  is  an  acute  febrile  affection  with  a 
tendency  to  stupor.  The  eruption,  which  appears  about  the  third  to  fifth  day,  is 
not  unlike  that  of  typhus  fever  and  tends  to  become  haemorrhagic.  Gangrene  oi 
penis  or  scrotum  may  appear.  It  is  transmitted  by  a  tick,  Dermacentor  andersoni 
(D.  venustus)  which  lives  on  domesticated  animals  of  the  region.  Destruction  of 
ticks  which  attach  themselves  to  sheep  by  dipping  has  been  proposed  as  a  measure 
for  eradication  of  the  disease.  Ricketts  found  that  the  reservoir  for  the  virus  is 
to  be  found  in  ground  squirrels,  chipmunks,  mountain  rats,  etc.,  and  that  ticks 
feeding  upon  them  become  infected  and  so  transfer  the  disease  to  man.  Guinea- 
pigs  are  susceptible  as  is  also  the  monkey.  Ricketts  noted  certain  chromatin  staining 
bacteria  in  man  and  in  eggs  of  infected  ticks  as  possibly  playing  a  part  in  etiology. 
Quite  recently  Wolbach  has  reported  the  finding  in  infected  guinea-pigs  of  organ- 
isms, possibly  bacterial,  showing  granular  and  lanceolate  forms.  They  are  par- 
ticularly abundant  in  the  endothelial  cells  of  blood-vessels.  They  are  from  ^  to 
i  micron  long  by  about  J^  micron  broad.  Ricketts  stated  that  his  organisms  were 
about  the  size  of  B.  influenza  and  showed  as  two  lanceolate-shaped  bodies.  Wilson 
and  Chowning,  in  1902,  reported  the  finding  of  piroplasm-like  organisms  in  the 
blood  of  the  disease.  Ricketts  proved  that  the  virus  was  not  filterable. 

Trachoma. — This  contagious  form  of  granular  conjunctivitis  is 
supposed  to  be  due  to  chlamydozoa  or  inclusion  bodies  and  is  classed 
as  one  of  the  filterable  viruses.  The  relation  of  the  trachoma  bodies 
to  the  Koch- Weeks  bacillus  is  discussed  under  that  organism. 


438  BERIBERI 

Typhus  Fever. — It  has  been  suggested  that  the  cause  may  be  a  pro- 
tozoon  transmitted  by  vermin. 

Recent  work  by  Anderson  and  Ricketts  has  shown  that  the  blood  of 
human  cases  is  infective  for  monkeys.  The  virus  does  not  seem  to 
pass  through  a  Berkefeld  filter  and  the  epidemiology  points  to  the 
body  louse  as  the  transmitting  agent.  Nicolle  reported  the  filter- 
ability  of  the  virus. 

Plotz  has  isolated  a  Gram-positive  pleomorphic  bacillus  from  the  blood  of  typhus 
patients  as  well  as  from  the  blood  of  guinea-pigs  and  monkeys  infected  by  injections 
of  typhus  blood.  It  is  most  abundant  in  blood  taken  four  or  five  days  before  the 
crisis.  It  only  grows  anaerobically  and  grows  best  in  ascitic  fluid  sterile  tissue  media. 
Morphologically  it  shows  curved,  straight  and  coccoid  forms.  The  rods  are  about  1.5 
micron  long.  The  serum  of  convalescents  shows  complement-fixation  bodies  as 
well  as  agglutinins.  The  organism  has  been  named  B.  typhi  exanthematici. 

Hort  states  that  only  blood  recently  taken  from  typhus  patients  will  cause  the 
disease  in  monkeys  while  the  same  blood  which  has  been  incubated  several  hours 
or  days  fails  to  produce  the  disease.  Others,  as  well  as  Hort,  doubt  the  etiologi- 
cal  relation  of  the  organism  of  Plotz  to  typhus  fever  or  the  mild  form  of  the 
disease  as  seen  in  New  York  City  and  there  known  as  Brill's  disease.  Tabardillo 
or  Mexican  typhus  is  the  same  as  typhus. 

Varicella. — Entirely  unknown. 

Whooping-cough.  —  Influenza-like  bacilli  have  been  implicated. 
Bordet-Gengou  bacillus. 

OF  TROPICAL  CLIMATES 

Ainhum. — A  disease  characterized  by  a  constricting  fibrous  ring, 
especially  of  little  toe,  often  leading  to  spontaneous  amputation. 

Beriberi. — Various  microorganisms  and  food  factors  suggested. 
A  form  of  multiple  neuritis,  occurring  chiefly  in  countries  where  rice 
is  the  staple  food,  characterized  by  oedema  and  marked  cardiac  and 
respiratory  embarrassment.  The  vagal  involvement  produces  grave 
symptoms.  Rice  from  which  the  pericarp  has  been  largely  removed, 
polished  rice,  implicated. 

Prior  to  the  investigations  of  Fraser  and  Stanton  the  importance  of  the  rice  factor 
in  the  etiology  of  beriberi  was  insisted  upon  by  Braddon  who  thought  that  a  poison 
was  elaborated  by  some  organism  which  poison  was  contained  in  the  beriberi  pro- 
ducing rice.  This  development  was  thought  to  occur  in  rice  stored  in  damp  places, 
but  Vedder  has  shown  that  storing  undermilled  rice  in  a  damp  place  for  a  year  does 
not  cause  it  to  lose  its  anti-beriberi  producing  properties.  The  work  of  Eijkman  in 
showing  that  polyneuritis  could  be  produced  in  fowls  by  feeding  them  on  polished 
rice  and  prevented  when  a  diet  of  rice  polishings  was  added  to  the  neutritis-produc- 


DISEASES  OF  UNKNOWN  ETIOLOGY  439 

ing  rice  opened  the  way  for  a  vast  amount  of  experimental  work.  As  regards  the 
nature  of  the  neuritis  preventing  substance  in  the  rice  polishings  it  was  soon  found 
that  it  had  no  relation  to  the  phosphorus  content.  Funk  has  isolated  a  substance 
he  calls  vitamine,  a  pyramidine  base  precipitated  by  phosphotungstic  acid,  which  is 
present  in  rice  in  the  proportion  of  i  to  100,000  and  seems  to  possess  extraordinary 
curative  properties  in  polyneuritis  gallinarum.  Heart  muscle,  egg  yolk  and  yeast 
are  rich  in  this  anti-neuritis  substance,  which  is  also  present  in  lentils  and  barley. 
Schaumann  considers  malt  as  richer  in  the  anti-neuritis  vitamine  than  any  other 
article  of  diet,  rice  bran  coming  next.  Many  think  that  vitamines  have  not  as  yet 
been  separated  but  that  they  are  intimately  combined  with  some  mother  substance 
in  the  food.  There  is,  in  all  probability,  a  large  number  of  vitamines  present  in 
various  animal  and  vegetable  foods,  the  deficiency  of  which  in  a  diet  may  lead  to 
vague  disorders  or  to  well-recognized  diseases,  such  as  scurvy,  ship-beriberi,  beriberi 
or  pellagra. 

Schaumann  considers  the  curative  principle  to  be  of  the  nature  of 
an  activator.  An  increase  in  the  ingestion  of  carbohydrates  and  nec- 
essarily in  the  vitamine  as  well  seems  to  produce  neuritis  more  rapidly 
than  where  a  smaller  amount  is  given,  this  indicating  the  importance 
of  these  vitamines  in  carbohydrate  metabolism. 

In  epidemics  of  beriberi  it  has  been  observed  that  those  who  eat  most  rice  are  more 
often  attacked,  thus  men  more  frequently  than  women.  A  temperature  of  1 2o°C.  de- 
stroys the  vitamine.  Owing  to  the  absence  of  rice  as  a  constituent  of  other  than  slight- 
est importance  in  the  dietary  of  Brazilian  cases  of  beriberi,  as  well  as  from  numerous 
reports  of  the  occurrence  of  the  disease  in  nonrice-eating  persons,  the  view  that  is 
now  entertained  is  that  not  only  polished  rice,  but  any  predominating  carbohydrate 
article  of  diet,  which  is  deficient  in  the  neuritis-preventing  substance,  can  produce 
beriberi.  Wellman  and  Bass  have  shown  that  such  articles  of  diet  as  sago,  boiled 
white  potatoes,  corn  grits  and  macaroni  practically  parallel  polished  rice  in  the  pro- 
duction of  polyneuritis  in  fowls. 

Blackwater  Fever. — Considered  as  a  malarial  disease,  but  thought  by 
some  to  be  possibly  caused  by  a  protozoon — a  Babesia  (Piro plasma). 
A  disease  usually  occurring  in  patients  with  a  malarial  history  and 
characterized  by  rapid  febrile  onset,  early  jaundice,  asthenia,  pain  in 
loins  and  the  pathognomonic  haemoglobinuria. 

Dengue. — Supposed  to  be  due  to  a  protozoon  transmitted  by  Culex 
fatigans.  A  disease  characterized  by  sudden  onset,  high  fever  for  three 
or  four  days,  pains  in  the  postorbital  regions,  back  and  about  joints. 
A  remission  occurs  on  the  third  to  fifth  day  followed  by  a  secondary 
rise  of  temperature  and  a  measles-like  eruption.  Leukopenia  and  re- 
duction in  the  percentage  of  polymorphonuclears.  Virus  exists  in 
the  blood  and  is  filterable. 


440  PELLAGRA 

Goundou. — Symmetrical  bony  tumors  of  nasal  processes  of  superior 
maxillary  bones. 

Oroya  Fever. — A  disease  with  a  fever  characterized  by  a  profound 
involvement  of  the  bone  marrow  producing  very  rapidly  an  anaemia 
resembling  that  of  pernicious  anaemia.  Pains  of  bones  and  joints 
marked.  See  Verruga. 

The  disease  is  chiefly  found  in  towns  situated  in  narrow,  wind-protected  valleys 
of  the  West  side  of  the  Andes,  at  elevations  of  from  3000  to  9000  feet.  Townsend 
has  suggested  that  a  species  of  Phlebotomus,  which  is  very  prevalent,  may  be  the 
transmitting  agent. 

Barton  isolated  a  paratyphoid  bacillus  from  the  blood  of  a  patient,  besides  which 
other  bacteria  have  also  been  isolated.  In  1909,  Barton  noted  certain  rod-like 
organisms  in  the  red  cells  of  Oroya  fever  patients  which  he  considered  protozoal  in 
nature. 

Strong  and  his  colleagues  found  in  the  blood  of  Oroya  fever  cases 
rod-shaped  forms  in  the  red  cells,  varying  from  i  to  2  microns  in  length, 
the  red  cells  containing  from  i  to  30  of  these  elements. 

Intravenous  inoculation  of  blood  containing  these  elements  into  monkeys  and 
rabbits  was  negative  in  result.  These  organisms  were  considered  as  intermediate 
between  bacteria  and  protozoa.  They  are  closely  related  to  Grahamella  and  the 
Harvard  commission  has  proposed  the  name  Bartondla  bacilliformis. 

Pellagra — This  disease  is  characterized  by  (i)  a  sprue-like  stomatitis 
and  disorders  of  alimentary  canal,  (2)  an  erythema  usually  limited  to 
parts  exposed  to  the  sun  and  characterized  by  marked  symmetry  and 
striking  delimitation  from  the  sound  skin  and  (3)  various  neurological 
manifestations  and  a  toxic  psychosis  which  may  go  on  to  confusional 
insanity.  The  disease  is  characterized  by  annual  recurrences  in  the 
spring  with  improvement  in  the  winter.  The  views  as  to  etiology 
which  consider  the  causative  agent  as  a  protozoon,  possibly  transmitted 
by  a  Simulium  or  by  Stomoxys  are  now  historical. 

The  maize  ideas  of  etiology  are  now  considered  as  having  a  bearing  only  in  con- 
nection with  the  larger  question  of  vitamine  deficiency  in  cereals  in  general.  Aless- 
andrini  has  recently  brought  forward  the  colloidal  silica  etiology. 

These  views  are  that  colloidal  silica  in  water  is  responsible  for  the  disease.  Voegt- 
lin  noted  the  great  amount  of  aluminium  in  certain  vegetables  and  suggested  this 
as  the  toxic  causative  substance.  A  mixture  of  colloidal  alumina  and  silica  in  water 
is  supposed  to  be  operative  as  well  as  silica  alone.  Against  the  colloidal  silica 
hypothesis  is  the  statement  of  Sandwith  that  the  water  of  the  Nile,  the  drinking 
water  of  Egypt,  is  low  in  colloidal  silica  content. 


DISEASES  OF  UNKNOWN  ETIOLOGY  441 

The  present  trend  of  thought  in  connection  with  pellagra  is  that  it 
is  a  food  deficiency  disease  connected  with  a  deficiency  in  vitamines 
necessary  for  normal  metabolism  (see  beriberi). 

In  February,  1915,  Goldberger  started  a  "pellagra  squad,"  consisting  of  n 
prisoners  on  a  diet  of  wheat  flour  (patent),  cornmeal,  corn  grits,  corn  starch,  polished 
rice,  granulated  sugar,  cane  syrup,  sweet  potatoes,  fat  fried  out  of  salt  pork,  cabbage, 
collards,  turnip  greens  and  coffee.  Baking  powder  was  used  for  making  biscuits 
and  corn  bread.  The  food  value  of  each  man's  diet  averaged  2952  calories. 

A  control  was  carried  out  with  prisoners  on  a  normal  diet.  The  experiment  was 
continued  until  Oct.  31,  1915.  Of  the  n  volunteers  on  the  excessive  carbohy- 
drate diet  six  developed  symptoms.  Loss  of  weight  and  strength  and  mild  neuras- 
thenia were  early  symptoms.  Definite  cutaneous  manifestations  appeared  only 
after  five  months.  The  skin  lesions  were  first  noted  on  the  scrotum,  later  appearing 
on  backs  of  hands  in  two  cases  and  back  of  neck  in  one  case. 

Just  as  with  rice  so  does  excessive  milling  of  wheat  get  rid  of  vitamines,  therefore 
bread  made  from  highly  milled  flour  is  dietetically  deficient. 

Again,  as  brought  out  by  Voegtlin,  alkalis  tend  to  destroy  any  remaining  vitamines 
in  such  bread.  The  practice  of  using  sodium  bicarbonate  in  preparation  of  bread 
is  a  further  factor  in  the  food  deficiency  problem.  With  the  use  of  baking  powder 
or  buttermilk  the  alkaline  carbonate  of  soda  is  neutralized  so  that  there  is  no  de- 
structive effect  on  vitamine  content. 

Notwithstanding  the  above  evidence  as  to  food  deficiency  etiology  it  must  be  re- 
membered that  McNeal  and  his  colleagues  on  the  Thompson-McFadden  pellagra 
commission,  as  well  as  other  authorities  on  this  disease,  insist  upon  a  probable 
infectious  agent  as  cause. 

Rat-bite  Disease. — A  disease  caused  by  the  bite  of  rats.  Rather 
common  in  Japan.  Five  weeks  after  bite  when  wound  has  healed, 
high  fever  sets  in,  cicatrix  becomes  inflamed  with  lymphangitis  and 
swollen  glands.  The  fever  falls  in  a  few  days  to  be  succeeded  by  other 
febrile  paroxysms.  An  erythematous  eruption  accompanies  the  second 
paroxysm. 

Supposed  by  Ogata  to  be  due  to  a  protozoon,  but  recent  work  by  Schotmuller, 
in  1914,  has  shown  that  the  cause  is  a  Streptothrix,  S.  muris  ratti.  This  finding  has 
been  corroborated  by  Blake.  The  organism  first  invades  the  lymphatic  structures 
and  then  the  blood,  giving  a  septicaemia.  Various  organs  are  later  involved.  Blake's 
case  developed  a  powerful  agglutinin  for  the  specific  Streptothrix. 

Sprue. — A  form  of  chronic  diarrhoea  characterized  by  diaphanous 
thinning  of  gut  and  ulcerations  of  buccal  cavity. 

Kohlbrugge  found  organisms  resembling  Oidium  albicans  in  the  intestines, 
oesophagus  and  tongue.  He  found  similar  organisms  in  the  stools  and  tongue 
scrapings  of  cases  of  sprue.  Beneke  found  bacilli  in  the  tongue,  oesophagus  and 
intestines  and  considered  these  as  causative,  regarding  the  thrush-like  membranous 
deposit  as  connected  with  the  cachectic  state  and  not  causative. 


442  YELLOW  FEVER 

Bahr  is  inclined  to  believe  that  Monilia  albicans  (Oidium  albicans}  is  the  cause 
as  he  found  these  saccharomycetes  in  the  deep  layers  of  the  tongue,  in  the  mucoid 
coating  of  the  intestines  and  in  the  deposit  in  the  oesophagus.  He  thinks  it  the 
ordinary  thrush  species  which  may  take  on  greater  virulence  in  the  tropics.  Ashford 
states  that  he  has  found  a  species  of  Monilia,  different  from  that  of  thrush,  almost 
constantly  in  tongue  scrapings  and  stools  of  sprue  cases  and  he  regards  this  species 
as  the  cause  of  sprue.  He  states  that  this  organism  is  common  in  Porto  Rico  bread 
and  thinks  it  possible  that  the  disease  is  transmitted  in  this  way.  Wood  has  re- 
cently expressed  the  view  that  sprue  is  not  infrequently  mistaken  for  pellagra  in 
the  southern  United  States. 

Tsutsugamushi. — A  disease  of  Japan  somewhat  resembling  typhus 
fever.  Supposed  to  be  due  to  a  protozoon  transmitted  by  the  Kedani 
mite. 

Verruga  Peruana. — A  disease  of  Peru  formerly  considered  as  a  later 
stage  of  Oroya  fever. 

The  eruption  of  verruga  somewhat  resembles  that  of  yaws  and  it  was  at  one  time 
suggested  that  verruga  was  simply  yaws  as  influenced  by  high  altitude.  Strong 
and  his  colleagues  found  that  they  could  infect  rabbits  intratesticularly  and  that 
lesions  resembling  those  of  man  could  be  produced  in  dogs  and  monkeys  by  cuta- 
neous and  subcutaneous  inoculations.  The  virus  has  been  transmitted  from  monkey 
to  monkey.  The  Wassermann  reaction  was  negative.  In  extracts  from  the  granu- 
lomatous  lesions  they  found  a  very  active  haemolysin.  It  will  be  remembered  that 
animals  are  not  susceptible  to  Oroya  fever  blood  inoculations. 

From  the  fact  that  it  is  possible  to  inoculate  a  person  by  rubbing  verruga  material 
on  a  scarified  surface  it  would  seem  that  the  infection  might  be  transmitted  by 
insects. 

Yellow  Fever. — Due  to  a  filterable  virus  transmitted  by  the  Stego- 
myia  calopus.  A  disease  characterized  by  sudden  onset,  rachialgia, 
albuminuria,  jaundice  and  often  haemorrhages  about  the  third  day. 
Pulse  becomes  slow  even  with  rising  temperature.  Black  vomit  often 

precedes  fatal  termination.     Virus  exists  in  the  blood  and  is  filterable. 
P 

The  virus  is  present  in  the  blood  of  the  peripheral  circulation  only  during  the 
first  three  days  of  the  disease.  A  female  Stegomyia  sucking  the  human  blood 
during  the  first  three  days  from  onset  of  fever  may  become  infected  but  cannot 
transmit  yellow  fever  to  a  second  person  until  after  the  expiration  of  at  least  twelve 
days,  during  which  time  some  development  of  the  virus,  of  the  character  of  which 
we  are  in  ignorance,  goes  on  in  the  mosquito. 

Seidelin  has  stated  that  he  has  found  a  protozoon,  Paraplasma 
flavigenum,  in  the  blood  of  yellow-fever  patients.  Authorities  generally 
deny  the  existence  of  this  parasite. 


APPENDIX 


A— PREPARATION    OF   TISSUES    FOR    EXAMINATION    IN 
MICROSCOPIC    SECTIONS 

Note.  The  most  important  step  in  the  preparation  of  sections  of  tissues  for  histological 
examination  is  proper  and  immediate  fixation.  This  step  in  the  technic  is  often 
in  the  hands  of  the  surgeon  at  the  time  of  the  operation  or  the  physician  at  autopsy 
and  it  should  be  understood  that  a  satisfactory  diagnosis  can  only  be  made  when 
the  pieces  of  tissue  are  at  once  dropped  into  a  fixative.  Various  protozoa,  as  amoebae, 
disintegrate  in  one  or  two  hours,  unless  properly  fixed  and  body  cells  show  degen- 
eration after  the  tissues  have  been  left  without  fixation  for  a  few  hours,  which 
changes  may  be  interpreted  as  pathological. 

Prepare  a  pint  or  quart  of  10%  formalin  solution  (4%  formaldehyde)  shortly 
before  operation  or  autopsy.  Drop  into  the  solution  slices  of  tissue,  not  more  than 
*y±  inch  thick,  as  soon  as  cut.  Leave  in  the  fixative  for  twenty-four  hours  and 
then  place  in  70%  alcohol.  The  pathologist  will  attend  to  the  other  steps. 

We  use  two  fixation  solutions  in  routine  work,  one  of  10%  formalin  and  one  of 
Zenker's  solution.  This  latter  requires  prolonged  washing  of  tissues  following 
fixation  and  has  little  advantage  over  formalin  for  ordinary  purposes. 

i.  Fixation: 

a.  It  is  most  important  that  the  tissues  to  be  examined  be  placed  in  the  fixing 
fluid  as  soon  after  death  or  operation  as  possible.     Degenerative  changes  are  in  this 
way  avoided. 

b.  The  piece  of  tissue  to  be  fixed  must  not  be  too  large.     Using  a  sharp  scalpel, 
or  preferably  a  razor,  a  slab  of  tissue  about  one-half  an  inch  square  and  not  more 
than  one-fifth  of  an  inch  thick  should  be  dropped  into  the  bottle  containing  the  fixa- 
tive.    The  bottom  of  this  bottle  should  have  a  thin  layer  of  cotton  with  a  piece  of 
filter-paper  covering  it.     There  should  be  at  least  20  times  as  great  a  volume 
of  fixing  fluid  as  of  tissue  to  be  fixed.     Delicate  tissues,  as  pieces  of  gut,  should  be 
attached  to  pieces  of  glass,  wood,  cardboard,  or  blotting  paper  before  being  placed  in 
the  fixative. 

c.  The  most  convenient  fixative  for  the  average  medical  man  is  (i)  a  10%  solution 
of  ordinary  commercial  formalin  (4%  of  formic  aldehyde  gas),  either  in  water  or, 
preferably,  in  normal  salt  solution.     Fixation  is  complete  in  from  twelve  to  twenty- 
four  hours.     By  placing  in  the  incubator,  at  37°C.,  two  to  twelve  hours  in  the  forma- 
lin solution  suffices.    If  fixed  in  the  paraffin  oven  (s6°C.),  fixation  is  accomplished 
in  about  one-half  hour. 

Formalin  once  used  for  fixation  must  be  thrown  away. 

(2)  The  fixative  which  probably  gives  the  best  histological  pictures  and  with  which 
we  obtain  the  most  satisfactory  haematoxylin  staining  is  Zenker's  fluid.  This  is 

443 


444  APPENDIX 

Miiller's  fluid  containing  5%  of  corrosive  sublimate.  It  also  contains  5%  of  glacial 
acetic  acid,  which  latter  is  only  added  just  before  we  are  ready  to  fix  the  piece  of 
tissue.  Muller's  fluid  is: 

Pot.  bichromate,  2 . 5  grams. 

Sod.  sulphate,  i .  o  grams. 

Water,  loo.oc.c. 

Zenker's  fluid  fixes  in  about  twenty-four  hours.  After  all  corrosive  sublima 
fixatives  we  should  wash  the  tissues  in  running  water  for  twelve  to  twenty-four 
hours.  The  precipitate  of  mercury  in  the  tissues  is  best  gotten  rid  of  by  treating  the 
section  on  the  slide  with  Lugol's  solution,  rather  than  the  tissue  in  bulk  with  iodine 
alcohol. 

(3)  In  Orth's  fluid  we  add  10%  of  formalin  to  Muller's  fluid  (recommended  for 
nerve  tissue). 

A  saturated  corrosive  sublimate  solution  in  salt  solution  with  the  addition  of  5% 
of  glacial  acetic  acid  may  be  used  as  a  substitute  for  Zenker's  fluid. 

(4)  Where  the  tissue  is  to  be  examined  chiefly  for  bacteria  absolute  alcohol  is  the 
best  fixative.     The  piece  of  tissue  should  be  small,  not  over  ^  inch  thick  and  sus- 
pended by  a  string  to  the  cork  so  as  not  to  lie  on  the  bottom  where  the  alcoholic 
strength  tends  to  become  weaker.     Better  histological  details  are  gotten  by  fixing 
for  two  hours  with  80%  alcohol  and  then  transferring  to  absolute  for  twelve  to 
twenty-four  hours. 

2.  Dehydration. — After  washing  for  twelve  to  twenty-four  hours  in  running 
water,  following  corrosive  sublimate  fixation,  or  simply  washing  for  a  few  minutes 
after  formalin,  the  tissues  should  be  placed  in  70%  alcohol.     They  may  be  kept  in 
this  indefinitely.    If  they  are  to  be  sent  to  a  laboratory  for  sectioning,  it  is  advisable 
to  moisten  a  pledget  of  cotton  in  70%  alcohol  and  fill  in  the  bottom  of  the  bottle 
with  it.    Then  drop  in  the  tissues  and  pack  in  gently  over  them  sufficient  70% 
alcohol-saturated  cotton  to  fill  up  the  bottle.     All  the  alcohol  should  be  absorbed 
by  the  cotton  so  that  if  the  bottle  should  break  in  transit  there  would  be  no  damage 
from  the  alcohol.     The  stopper  of  the  bottle  should  be  paraffined  or  sealed  with 
wax. 

Tissues  may  be  left  in  the  70%  alcohol  twelve  to  twenty-four  hours  and  should 
then  be  transferred  to  95%  alcohol  for  an  equal  time.  They  are  then  transferred  to 
absolute  alcohol,  where  they  remain  from  two  to  twelve  hours  and  are  then  placed  in 
xylol.  The  time  in  xylol  should  be  as  short  as  possible.  So  soon  as  the  tissue  looks 
clear  it  should  be  removed — thirty  minutes  to  two  hours. 

Bolles  Lee  is  a  strong  advocate  of  the  superiority  of  cedar  oil  over  xylol  or  any  other 
clearing  agent  for  paraffin  imbedding.  It  does  not  affect  delicate  structures  nor  make 
them  brittle  even  when  kept  in  the  cedar  oil  for  weeks  or  months.  Furthermore, 
it  does  not  matter  whether  the  cedar  oil  is  entirely  gotten  rid  of  before  sectioning  the 
paraffin  as  is  the  case  for  best  results  with  xylol.  Cedar  oil  will  clear  from  95% 
alcohol  as  well  as  from  absolute  alcohol. 

3.  Imbedding. — The  tissue  is  now  transferred  to  melted  paraffin.     Paraffin  melt- 
ing at  48°C.  for  winter  work,  and  that  melting  at  54°C.  for  summer  is  to  be  recom- 
mended.    The  time  in  the  paraffin  should  not  be  prolonged.     Two  hours  will 
ordinarily  suffice.     Some  leave  in  the  paraffin  for  twelve  to  twenty-four  hours. 


APPENDIX  445 

Next  take  a  paper  box  (made  of  stiff  writing-paper  folded  over  a  square  of  wood) 
and  fill  with  the  melted  paraffin.  As  quickly  as  possible  drop  in  the  piece  of  tissue 
taken  out  of  the  paraffin  bath  with  heated  forceps  and,  so  soon  as  the  paraffin  begins 
to  solidify  on  the  surface,  place  the  paper  box  in  ice  water.  When  paraffin  is  rapidly 
cooled,  crystallization  is  less. 

The  Acetone  Method. — Take  the  tissues  out  of  the  70%  alcohol  and  place  in  ace- 
tone. After  remaining  in  acetone  for  one  or  two  hours,  the  tissues  should  be  trans- 
ferred to  fresh  acetone  for  an  equal  length  of  time.  Dry  calcium  chloride  in  the  bot- 
tom of  the  acetone  bottles  keeps  it  dehydrated.  They  should  then  be  placed  in 
xylol  for  about  one-half  hour  and  then  embedded  in  paraffin  as  directed  above. 

The  Chloroform  Method. — The  procedure  may  be  the  same  as  in  the  method  of 
passing  through  alcohols  to  xylol,  substituting  chloroform  for  xylol  and  then  trans- 
ferring to  paraffin. 

Where  absolute  alcohol  is  not  obtainable,  very  satisfactory  results  may  be 
obtained  by  transferring  tp  a  mixture  of  95%  alcohol  and  chloroform  after  immer- 
sion in  95%  alcohol.  Then  going  from  the  alcohol-chloroform  mixture  to  pure 
chloroform,  thence  to  paraffin. 

Rapid  paraffin  imbedding  methods. 

When  a  piece  of  tissue  is  not  more  than  Y±  inch  square  and  ^  inch  thick,  it  is 
very  easy  to  run  it  through  in  three  to  six  hours.  Thus: 

10%  Formalin  (in  37°C.  incubator),  i    hour. 

70%  Alcohol  (in  37°C.  incubator),  i    hour. 

95%  Alcohol  (in  37°C.  incubator),  i    hour. 

Absolute  alcohol  (in  37°C.  incubator),  H  hour. 

Xylol  (in  37°C.  incubator),  V?.  hour. 

Paraffin  (in  55°C.  incubator),  ^  to  2  hours. 

Method  of  Lubarsch. — In  this  excellent  method  small  pieces  of  tissue  not  more 
than  }/$  inch  thick  are  placed  in  a  wide  test-tube  containing  10%  formalin  for  10  to 
15  minutes,  changing  the  fluid  twice.  Transfer  to  95%  alcohol  10  minutes  changing 
alcohol  once.  Absolute  alcohol,  for  10  minutes  changing  twice.  Pure  aniline  oil 
until  tissues  are  transparent,  15  to  30  minutes.  Xylol,  changing  two  to  three  times 
or  until  the  xylol  is  no  longer  yellow,  10  to  20  minutes.  Imbed  in  paraffin  for  20 
minutes  to  i  hour.  During  the  entire  process  keep  the  test-tube  in  a  water  bath 
or  incubator  at  5o°C.  It  is  necessary  to  have  a  good  microtome.  The  best  is  that 
of  Minot.  Very  satisfactory  sections  can  be  cut  with  the  various  types  of  student 
microtomes,  costing  from  twelve  to  twenty  dollars. 

(In  using  a  hand  microtome,  a  razor  with  a  flat  edge  is  necessary.  After  expe- 
rience, sections  thin  enough  for  histological  but  not  for  bacteriological  examination 
can  be  made.) 

If  the  piece  of  tissue  is  properly  dehydrated  and  imbedded,  thin  sections  (3  to 
ioju)  should  be  easily  obtained,  provided  the  knife  be  sharp.  One  advantage  about 
the  paraffin  method  is  that  it  is  only  necessary  to  have  a  small  part  of  the  blade  in 
proper  condition.  With  celloidin  the  entire  cutting  edge  must  be  perfect.  Having 
cut  the  sections,  they  should  be  dropped  on  the  surface  of  a  bowl  of  warm  water 
(45°C.).  This  causes  the  section  to  flatten  out  evenly. 

Decakification.—This  is  best  accomplished  by  fixing  in  10%  formalin  for  twenty- 


446  APPENDIX 


:h  square 


four  hours,  then  placing  a  small  piece  of  the  bone  (not  exceeding  ^  inch  sqi 
and  %  inch  thick)  in  concentrated  sulphurous  acid. 

This  decalcifies  in  about  two  to  seven  days.  Wash  thoroughly  in  alkaline  water 
and  then  in  tap  water.  Pass  through  alcohols  and  xylol  and  imbed  and  section  as 
before  described. 

To  Stain  Sections. — It  is  first  necessary  to  affix  the  section  to  a  slide  or  cover-glass 

To  attach  the  section  firmly  to  the  slide,  so  that  it  will  not  become  detached  in 
subsequent  treatment,  pick  up  a  section  on  a  strip  of  cigarette  paper. 

A  sheet  of  cigarette  paper  is  cut  into  about  five  pieces  (%  X  iM  inch).  In- 
serting the  strip  of  cigarette  paper  under  the  section,  it  is  easily  lifted  up  out  of 
the  water.  Then  apply  the  slip  of  cigarette  paper,  section  downward,  to  a  perfectly 
clean  slide.  Blot  with  a  piece  of  filter-paper,  then  strip  off  the  piece  of  filter-paper 
leaving  the  section  smoothly  applied  to  the  slide.  Next  place  in  the  37°C.  incubator 
for  twelve  to  twenty-four  hours  and  the  section  will  be  found  to  be  so  firmly  attached 
that  it  will  not  be  dislodged  by  subsequent  treatment. 

For  Immediate  Diagnosis. — Take  a  very  small  loopful  of  albumin  fixative  (white 
of  fresh  egg,  50  c.c.,  glycerine,  50  c.c.;  sodium  salicylate,  i  gram)  and  deposit  it  on  a 
cover-glass.  Now  take  up  a  loopful  of  30%  alcohol  (i  drop  of  95%  alcohol  and  2 
drops  of  water)  and  applying  it  over  the  albumin  fixative,  smear  out  the  mixture 
uniformly  over  the  cover-glass. 

2.  Pick  up  a  section  on  a  strip  of  cigarette  paper  and  apply  it  to  the  prepared  sur- 
face on  the  cover-glass.     Blot  with  gentle  pressure  with  a  piece  of  filter-paper  over 
the  strip  of  cigarette  paper,  and  strip  off  this  latter,  leaving  the  section  attached  to 
the  cover-glass. 

3.  Now,  turning  the  flame  of  the  Bunsen  burner  down  very  low  or  with  a  small 
alcohol  flame,  we  hold  the  cover-glass  in  a  Stewart's  forceps,  section  side  up,  over 
the  flame  and  slowly  lower  it  until  the  paraffin  is  observed  to  melt.     This  shows  a 
temperature  of  about  5o°C.     The  section  is  fixed  by  the  coagulation  of  the  albumin 
at  about  7o°C.     To  obtain  this  temperature  lower  the  cover-glass  still  more,  and  the 
moment  vapor  is  seen  to  rise  from  the  section  it  indicates  the  attachment  of  the  sec- 
tion to  the  cover-glass. 

4.  Flood  section  on  cover-glass  or  slide  with  xylol;  this  dissolves  out  the  paraffin. 
It  is  better  to  pour  off  the  first  xylol  and  drop  on  fresh  xylol  (one  minute). 

5.  Remove  xylol  with  two  applications  of  absolute  alcohol  (one  minute). 

6.  Treat  specimen  with  two  or  three  applications  of  95%  alcohol  (one  to  two 
minutes). 

7.  Next  wash  in  water  (one  to  two  minutes). 

8.  Flood  specimen  with  haemalum  or  Delafield's  haematoxylin  (three  to  seven 
minutes). 

9.  Wash  in  tap  water  for  about  two  to  five  minutes  until  a  purplish  tinge  is  devel- 
oped hi  the  section.     The  alkali  in  ordinary  tap  water  develops  this  color. 

10.  Apply  i  to  1000  eosin  (aqueous)  for  thirty  seconds  to  one  minute. 

11.  Wash  in  water;  then  in  95%  alcohol;  then  in  absolute  alcohol. 

12.  Apply  a  few  drops  of  xylol  and  as  soon  as  the  section  is  perfectly  transpa 
mount  in  balsam,  or  immersion  oil. 

The  staining  by  haematoxylin  and  eosin  is  the  best  for  the  study  of  the  histology 
of  a  section.  It  only  requires  about  ten  minutes  to  run  a  preparation  through  for 
diagnosis  by  this  method. 


APPENDIX  447 

The  reagents  are  best  kept  in  dropping-bottles. 

The  staining  of  sections  on  slides  is  exactly  as  for  those  on  cover-glasses.  Cop- 
lin's  staining  jars  are  very  convenient  for  use  in  staining  slides. 

Where  the  cover-glass  method  is  used,  staining  by  Gram's  method,  acid-fast  stain- 
ing, capsule  staining,  etc.,  may  be  carried  out  as  for  bacterial  preparations. 

For  staining  Gram-positive  bacteria  in  sections,  the  Gram  method  as  for  bacterial 
preparations,  using  dilute  carbol  fuchsin  as  a  counterstain,  gives  good  results. 

For  Gram-negative  bacteria  stain  with  thionin  as  for  blood  preparations  (ten  to 
twenty  minutes).  Then  differentiate  in  i  to  500  acetic  acid  solution  for  ten  to 
twenty  seconds,  wash  with  water,  then  with  95%  alcohol,  and  quickly  through  abso- 
lute alcohol  and  xylol. 

Nicolle's  Method.— i.  Stain  with  Loffler's  methylene  blue  ten  to  fifteen  minutes. 

2.  Differentiate  in  i  to  500  acetic  acid  ten  to  twenty  seconds. 

3.  Place  in  i%  solution  of  tannin  for  a  few  seconds  (fixes  color). 

4.  Wash  in  water,  then  into  95%  alcohol,  absolute  alcohol,  xylol,  and  balsam. 

WEIGERT'S   IRON   ILEMATOXYLIN 

Solution  I 

Haematoxylin,  i  gram. 

Alcohol  (95%),  100  c.c. 

This  must  be  allowed  to  ripen  for  some  days  and  does  not  keep  over  six  months. 

Solution  H 

Liq.  Ferri  sesquichlor.  sp.  gr.  1.124  (about  10%)  4  c.c. 

HCL,  i  c.c. 

Water,  100  c.c. 

Mix  equal  parts  of  number  one  and  number  two.  The  mixture  only  keeps  about 
three  days.  The  HC1  prevents  overstaining. 

This  stain  followed  by  Van  Giesen's  stain  gives  more  perfect  results  than  any 
common  method  of  staining.  The  iron  haematoxylin  intensifies  the  sharpness  of 
the  Van  Geisen  differentiation. 

Other  iron  haematoxylin  stains  are  given  under  staining  for  protozoa. 

Van  Giesen's  Stain. — Take  of  i%  aqueous  solution  acid  fuchsin  from  5  to  15 
c.c.  Saturated  aqueous  solution  picric  acid  100  c.c.  The  method  of  using  is  to  first 
stain  with  haematoxylin  in  the  usual  way.  Then  pour  on  the  picric-acid  fuchsin 
solution  and  allow  to  stain  for  one  to  five  minutes.  Wash,  pass  through  alcohols 
and  xylol  and  mount  in  balsam. 

Connective-tissue  fibers,  axis  cylinders,  and  ganglion  cells  are  stained  a  bright 
garnet  red.  Myelin,  muscle  fibers,  and  cells  generally  are  stained  yellow.  Nuclear 
staining  is  that  of  haematoxylin.  The  stronger  stain  is  used  for  nerve  tissue;  the 
weaker,  for  demonstrating  connective  tissue  in  tumors. 

Levaditi's  Method. — Take  small  pieces  of  tissue,  about  2  mm.  in  thickness,  and 
harden  in  10%  formalin  for  twenty-four  hours  and  then  in  alcohol  for  the  same  pe- 
riod; then  wash  in  water  for  a  short  period.  They  are  stained  in  a  freshly  made  solu- 
tion of  silver  nitrate  1.5%  for  three  successive  days,  changing  the  solution  each  day, 


448  APPENDIX 

maintaining  the  blood  temperature,  and  excluding  light.  The  tissue  is  then  ph 
in  a  2%  solution  of  pyrogallic  acid,  with  the  addition  of  5%  formalin.  After  remain- 
ing in  this  for  twenty-four  hours,  light  being  excluded,  they  are  passed  through 
85%,  95%,  and  absolute  alcohol,  respectively;  embedded  in  paraffin;  and  cut  in 
about  5  micron  sections.  Equally  good  results  may  be  obtained  by  allowing  the 
silver  nitrate  to  act  at  room  temperature  and  embedding  in  celloidin. 

Nogitchi  has  used  the  following  modification  in  demonstrating  spirochaetes  in 
brain  and  cord:  Fix  ^-inch  slabs  of  tissue  in  10%  formalin  for  four  or  five  days. 
Then  place  tissues  in  the  following  solution:  Formalin  10  c.c.,  pyridin  10  c.c., 
acetone  25  c.c.,  absolute  alcohol  25  c.c.,  distilled  water  30  c.c.  Keep  in  this 
solution  for  five  days  at  room  temperature.  Then  wash  in  water  for  one  day. 
Transfer  to  95%  alcohol  for  three  days  and  then  wash  in  water  for  one  day.  Put 
tissue  in  dark  bottle  in  i^%  aqueous  solution  of  silver  nitrate  for  five  days  at  room 
temperature.  Wash  in  distilled  water  for  five  to  six  hours.  Transfer  to  reducing 
mixture  of  95  c.c.  of  4%  aqueous  solution  of  pyrogallol  and  5  c.c.  of  formalin.  Keep 
in  this  solution  twenty-four  hours.  Wash  in  water  and  put  through  alcohol  and 
xylol.  Imbed  in  paraffin. 

Romanowsky. — Staining  sections  with  Romanowsky  stains  is  not  very  satis- 
factory. The  differential  staining  seems  to  fade  out  in  passing  through  the  alcohols. 
This  may  be  avoided  by  blotting  the  section  after  staining  and  differentiation  and 
then  applying  the  xylol  to  the  blotted  section.  After  staining  with  Giemsa's  stain 
for  ten  to  fifteen  minutes,  differentiate  with  i  to  500  acetic  acid.  When  the  section 
has  a  pinkish  tinge,  wash  in  water,  dry,  clear  in  xylol,  and  mount. 

Good  tissue  staining  may  be  gotten  with  Wright's  stain.  After  removing  the 
paraffin  with  xylol  and  the  xylol  with  absolute  alcohol,  pour  on  a  sufficient  number  of 
drops  of  stain  and  after  one  minute  dilute  with  an  equal  number  of  drops  of  water. 
Allow  the  diluted  stain  to  remain  for  three  to  five  minutes.  Next  wash  in  water, 
differentiate,  until  the  tissue  has  a  pinkish  tinge,  in  i  to  500  acetic  acid.  This 
differentiation  is  best  done  in  a  tumbler  of  the  dilute  acetic  acid. 

After  washing  in  water,  quickly  pass  through  95%  and  absolute  alcohol,  clear  in 
xylol,  and  mount. 

I  now  use  the  panoptic  method  for  staining  tissues.  In  this  I  stain  with  Wright's 
stain  as  given  above  but  following  the  washing  of  the  section  we  treat  this  with  a 
dilute  Giemsa  (1-15)  for  ten  to  fifteen  minutes.  Then  wash  and  differentiate  in  i  to 
1000  acetic  acid  in  water  in  a  small  beaker.  When  the  section  assumes  a  pinkish 
tinge  wash  in  tap  water,  then  in  95%  and  absolute  alcohol  and  clear  in  xylol. 
Then  mount  in  liquid  petrolatum,  immersion  oil  or  balsam. 

Skin  Sectioning. — Of  all  tissues  that  of  skin  offers  the  greatest  difficulty  in  pre- 
paring sections.  The  best  results  can  probably  be  obtained  by  fixation  in  picro- 
sublimate  (saturated  aqueous  solution  picric  acid  i  part;  saturated  aqueous  solu- 
tion bichloride  of  mercury  one  part);  to  this  stock  mixture  add  5%  glacial  acetic 
acid  just  before  using.  Fix  small  pieces  of  skin  six  to  eighteen  hours.  Transfer 
direct  to  70%  alcohol  in  which  the  tissue  may  be  kept  indefinitely. 

For  sectioning  run  through  alcohols  to  absolute  and  then  to  a  mixture  of  absolute 
alcohol  and  carbon  bisulphide  (equal  parts).  Leave  until  tissue  sinks,  then  transfer 
to  pure  carbon  bisulphide  until  tissue  sinks.  Then  transfer  to  a  saturated  solu- 
tion of  paraffin  in  carbon  bisulphide  and  thence  to  paraffin.  Bisulphide  of  carbon 


APPENDIX 


449 


has  the  disadvantage  of  foul  odor  and  inflammability  but  does  not  seem  to  render 
tissues  brittle  and  difficult  to  section  as  does  xylol. 

NEUROLOGICAL  STAINING  METHODS 

Neuropathology  practically  dates  from  the  introduction  of  Marchi's  method  of 
staining  in  1885. 

Ordinary  osmic  acid  stains  both  normal  and  pathological  fat.  With  Marchi's 
method  only  the  oleic  acid  of  fatty  degeneration  is  stained. 

The  method  is  not  useful  until  three  or  four  days  have  elapsed  from  the  onset  of  the 
condition  causing  the  degeneration  and  it  is  applicable  for  only  three  or  four  months 
because  by  that  time  phagocytes  have  taken  up  the  pathological  fat  which  is  stained 
in  the  Marchi  method.  The  Weigert  method  is  the  one  to  use  after  a  period  of  three 
or  four  months.  In  Weigert's  stain  only  the  normal  myelin  sheath  is  stained  and  the 
lack  of  staining  of  myelin  sheaths  in  degenerated  areas  is  the  basis  of  the  stain. 
For  demonstrating  axonal  reactions  or  other  degenerative  changes  in  nerve-cells,  as 
shown  by  bulging  of  the  concave  sides  of  the  cells,  eccentric  nucleus  and  granular 
appearance  of  the  tigroid  bodies,  Nissl's  method  is  the  best. 

For  neuroglia  fiber  staining  Mallory's  phosphotungstic  acid  haematoxylin  is  to 
be  recommended. 

I.  For  Marchi's  Method.— Small  pieces  of  nerve  tissue  are  hardened  in  Miiller's 
fluid  for  seven  to  ten  days  and  are  then  transferred  to  a  mixture  of  two  parts  Muller's 
fluid  and  one  part  of  a  i%  osmic  acid  solution  and  should  remain  in  this  mixture 
for  about  seven  days.     The  tissue  thus  treated  is  run  through  alcohols  and  imbedded 
in  paraffin  in  the  usual  way. 

II.  For  Weigert-Pal  Method. — Thin  slices  of  tissue  are  fixed  in  10%  formalin 
in  about  four  days.     The  tissue  should  then  be  transferred  to  5  %  potassium  bichro- 
mate for  about  twelve  days.     The  tissue  is  then  imbedded  and  sections  cut.     If 
only  recently  mordanted  these  sections  may  be  at  once  stained  with  Weigert's 
haematoxylin  for  twelve  to  twenty-four  hours  (10  c.c.  ripened  10%  solution  haema- 
toxylin in  absolute  alcohol  and  90  c.c.  water).     Wash  in  water  to  which  about  2% 
of  a  saturated  solution  of  lithium  carbonate  has  been  added.    Now  differentiate 
from  one-half  to  five  minutes  in  %%  solution  of  potassium  permanganate  until 
the  gray  matter  looks  a  brownish  yellow.     Next  treat  sections  with  oxalic  acid 
i  gram,  potassium  sulphate  i  gram  and  water  200  c.c.  until  the  gray  matter  is 
almost  colorless.     This  takes  only  a  few  seconds.     Wash  in  water,  pass  through 
alcohols  and  xylol  and  mount  in  balsam. 

III.  For  Nissl  staining  either  thionin  or  Giemsa  staining  is  satisfactory. 

IV.  For  neuroglia  fiber  staining  use  Mallory's  Phosphotungstic  acid  haematoxylin. 

Take  of  haematein  ammonium,  o .  i  gram. 

Water,  loo.oc.c. 

Phosphotungstic  acid  crystals  (Merck)  2 .  o  grams. 

Dissolve  the  haematein  in  a  little  water  with  the  aid  of  heat,  and  add  it  after  it  is 
cool  to  the  rest  of  the  solution;  no  preservative  is  required.  If  the  solution  stains 
weakly  at  first,  it  may  be  ripened  by  the  addition  of  5  c.c.  of  a  Y±%  aqueous 
solution  of  potassium  permanganate,  or  it  may  be  allowed  to  stand  for  a  few  weeks 
until  it  ripens  spontaneously. 
29 


450  APPENDIX 

MAKING  AND  STAINING  OF  FROZEN  SECTIONS 

The  various  types  of  ether  freezing  microtomes  are  not  very  satisfactory  when 
only  used  occasionally.  With  the  general  introduction  of  cylinders  containing 
compressed  carbon  dioxide,  which  is  used  for  aerating  waters,  we  have  at  hand  a 
practical  and  convenient  method  of  making  frozen  sections. 

The  instrument  makers  furnish  a  freezing  microtome  of  the  Bardeen  type  whicl 
can  be  attached  directly  to  the  cylinder  by  a  revolving  clamp  nut. 

It  is  necessary  to  have  a  stand  to  support  the  iron  cylinder  in  a  horizontal  posi- 
tion. The  tissue,  which  may  be  taken  at  operation  for  immediate  diagnosis,  or 
which  preferably  has  been  fixed  in  formalin  for  twelve  to  eighteen  hours  is  immedi- 
ately placed  in  water.  If  the  tissues  have  been  in  alcohol  it  will  require  hours  of 
washing  before  they  can  be  frozen.  The  piece  of  tissue  which  is  to  be  frozen  should 
not  be  more  than  one-fifth  of  an  inch  thick.  Having  placed  the  piece  of  tissue  on 
the  freezing  box  of  the  microtome  we  turn  the  valve*  of  the  cylinder  to  allow  the 
gradual  escape  of  gas.  When  frozen  solid,  we  elevate  the  freezing  box  holding  the 
frozen  tissue,  by  revolving  a  graduated  disk  with  the  left  hand.  In  the  right  hand 
we  firmly  grasp  a  well-sharpened  blade  of  a  carpenter's  plane  mounted  in  a  wooden 
handle.  This  is  held  at  an  angle  of  45  degrees  to  the  polished  ways  of  the  micro- 
tome. By  alternate  shoving  and  withdrawing  of  the  blade,  held  rigidly,  we  accumu- 
late on  the  blade  a  number  of  sections.  Then  dip  the  blade  in  a  vessel  of  water  to 
detach  the  sections  which  float  in  the  water.  Keep  repeating  the  process  until 
numerous  satisfactory  sections  are  obtained.  Handles  for  holding  the  Gillette 
razor  blades  are  good  substitutes  for  the  carpenter's  plane. 

These  sections  may  be  picked  up  with  a  strip  of  cigarette  paper  and  applied  to  a 
clean  slide  upon  which  a  very  small  loopful  of  albumin  fixative  has  been  smeared 
out  with  30%  alcohol.  The  piece  of  cigarette  paper  with  the  section  underneath 
is  then  firmly  smoothed  out  upon  the  slide  with  filter-paper.  The  piece  of  cigarette 
paper  is  then  carefully  stripped  off  and  the  section  remains  attached  to  the  slide. 
By  careful  heating  over  a  very  small  flame,  until  the  vapor  just  arises,  the  section 
is  fixed  to  the  slide  and  we  can  then  stain  the  section  in  any  way  that  may  be  desired. 

NOTE. — The  procedures  for  carrying  the  tissues  through  celloidin  are  not  given 
as  it  requires  perfect  condition  of  the  entire  cutting  surface  of  the  microtome  knife 
and  a  considerable  time  for  the  passage  through  reagents  and  celloidin.  It  is  more 
suitable  as  a  method  when  sections  for  class  work  are  to  be  prepared. 

B— MOUNTING  AND  PRESERVATION  OF  PATHOLOGICAL  SPECIMENS 
AND  ANIMAL  PARASITES 

To  Mount  Small  Round  Worms. — Wash  the  hook,  whip,  or  filarial  worm  in  salt 
solution,  then  drop  in  70%  alcohol  containing  5%  of  glycerine;  the  glycerine-alcohol 
mixture  being  at  a  temperature  of  6o°C.  When  cool,  pour  into  Petri  dishes  and 
allow  the  alcohol  to  evaporate  in  the  37°C.  incubator. 

Mount  in  glycerine  jelly,  preferably  in  a  concave  slide,  and  ring  the  preparation 
with  gold  size.  The  following  is  the  formula  for  Kaiser's  glycerine  jelly:  Soak  one 
part  of  gelatin  in  six  parts  of  distilled  water  for  two  hours.  Then  add  seven  parts 
of  glycerine.  To  the  mixture  add  i%  of  carbolic  acid,  warm  for  fifteen  minutes, 
with  constant  stirring,  and  then  filter  through  cotton. 


APPENDIX 


451 


To  Prepare  Tape-worms. — Wash  in  salt  solution.  Wrap  around  a  piece  of  glass 
as  a  glass  slide  and  fix  in  salt  solution  containing  2  to  5%  of  formalin.  Then  keep 
the  preparation  permanently  in  70%  alcohol.  If  preferred,  the  specimen  may  be 
run  through  alcohols  and  xylol  and  mounted  in  balsam. 

Larvae. — Mosquito  larvae  may  either  be  prepared  as  for  small  round  worms  or 
they  may  be  dropped  into  70%  alcohol  at  6o°C.  and  then  passed  through  alcohols 
and  cleared  in  xylol  and  mounted  in  balsam.  Flukes  and  insects  may  require  treat- 
ment with  hot  (60°  to  7o°C.)  solution  of  10  to  20%  sodium  hydrate  solution.  Then 
wash  thoroughly  in  water  and  subsequently  pass  through  alcohols  to  xylol  and  mount 
in  balsam.  Clove  oil  or  cedar  oil  clears  more  slowly,  but  makes  specimens  less  brit- 
tle than  does  xylol.  Another  satisfactory  method  is  to  drop  insects  or  larvae  into 
acetone  at  6o°C.  and  after  being  in  this  from  one  to  twelve  hours  to  clear  in  xylol 
or  clove  oil  and  mount  in  balsam. 

Nematodes. — Looss  has  a  method  of  first  washing  a  small  nematode  or  delicate 
fluke  in  salt  solution.  Then  pouring  this  first  salt  solution  out  of  the  test-tube  in 
which  the  washing  was  carried  out,  to  add  fresh  salt  solution,  and  then  an  equal 
amount  of  saturated  aqueous  solution  of  bichloride  of  mercury.  The  shaking  is 
easily  carried  on  in  the  test-tube.  After  washing  in  water  the  worm  is  passed 
through  alcohols,  one  strength  of  which  should  contain  iodine.  Clear  in  xylol  and 
mount  in  balsam. 

An  excellent  method  is  that  of  Langeron. 

After  washing  in  salt  solution  fix  for  a  few  hours  in  5%  formalin.  Then  transfer 
to  lactophenol  which  has  been  diluted  with  an  equal  amount  of  water.  Allow  to 
remain  in  this  solution  for  several  hours  and  then  transfer  to  pure  lactophenol  in 
which  fluid  the  specimens  are  to  be  mounted.  Ring  with  paraffin  or  with  gold  size. 
(To  make  lactophenol  take  2  parts  of  glycerine  and  i  part  each  of  distilled  water, 
crystallized  carbolic  acid  and  lactic  acid.) 

A  quick  method  of  preparing  small  nematodes  for  examination  is  to  fix  them  for 
from  two  to  twelve  hours  in  5  to  10%  formalin,  this  being  heated  at  6o°C.  at  the 
time  the  worms  are  dropped  into  it.  Then  transfer  to  the  following  solution: 

Glucose  syrup  (glucose,  48;  water,  52),  100  c.c. 

Methyl  alcohol,  20  c.c. 

Glycerine,  10  c.c. 
Camphor,  q.s.  (a  small  lump  for  preservation). 

They  may  be  mounted  directly  in  this  and  the  cover-slip  ringed  with  about  6o°C, 
paraffin,  followed  with  gold  size. 

Preparations  so  cleared  and  mounted  in  glycerine  jelly  should  also  be  ringed  with 
paraffin  or  some  cement. 

Flukes,  cestodes,  and  nematodes  are  best  stained  with  carmine.  The  following 
is  a  good  formula. 

Dissolve,  by  boiling,  4  grams  carmine  in  30  drops  HC1  and  15  c.c.  water.  Then 
add  95  c.c.  of  85%  alcohol  and  filter  while  hot.  Neutralize  with  ammonia  until 
precipitate  begins  to  form.  Then  filter  cold. 

i.  Stain  parasites  taken  from  70%  alcohol  for  five  to 'twenty  minutes.  2.  Dif- 
ferentiate in  3%  hydrochloric  acid.  3.  Pass  through  alcohols  to  xylol  and  mount  in 
balsam. 


452 


APPENDIX 


Mites,  Fleas  and  Various  Small  Insects. — By  simply  taking  i  or  2  drops  of 
liquid  petrolatum  and  mounting  the  specimen  in  it  then  covering  with  a  cover- 
glass  one  is  able  to  study  the  details  of  these  objects  almost  as  well  as  if  they  were 
passed  through  acetone  and  xylol  into  balsam.  Liquid  petrolatum  is  also  most 
excellent  for  mounting  the  aerial  hyphae  of  fungi  with  their  sporangia  as  well  as  for 
Romanowsky  stained  blood  smears. 

Pathological  Specimens. — Pathological  tissues  which  are  to  be  sent  to  a  labora- 
tory for  sectioning  or  to  be  kept  for  future  study  should  be  fixed  by  one  of  the 
methods  given  in  Section  A  of  the  appendix. 

Formalin  fixation  is  the  more  convenient — that  with  Zenker's  fluid  the  more 
perfect.  After  fixation  with  Zenker's  fluid  the  pieces  of  tissue  must  be  washed  in 
running  water  over  night. 

After  fixation  the  pieces  of  tissue  are  transferred  to  70%  alcohol  in  which  they  ma) 
be  kept  indefinitely. 

For  preservation  of  gross  specimens  the  method  of  KAISERLING  is  generally  use< 

Fix  for  from  one  to  five  days  in  Solution  i. 

Solution  I 

Formaldehyde,  200  c.c. 

Water,  1000  c.c. 

Nitrate  of  potassium,  15  grams. 

Acetate  of  potassium,  30  grams. 

The  position  of  the  specimen  should  be  changed  from  day  to  day.  There  must 
be  at  least  five  times  as  much  fluid  as  specimen.  Drain  and  transfer  to  80%  alcohol 
for  a  few  hours,  then  into  95%  alcohol  until  the  natural  color  is  just  restored. 

Finally  preserve  in 

Acetate  of  potassium,  200  grams. 

Glycerine,  400  c.c. 

Water,  2000  c.c. 

It  is  advisable  to  keep  these  specimens  in  the  dark  as  light  destroys  the  natui 
color. 

To  Prepare  Flies  or  Mosquitoes  for  Transmission  through  the  Mails.— Wrap 
the  insect  carefully  in  a  piece  of  tissue  paper  (toilet-paper  answers).  Impregnate 
sawdust  with  5%  carbolic  acid  solution  and  fill  around  the  tissue  paper  in  the  box 
containing  them.  (Barely  moisten.) 

It  is  very  satisfactory  to  take  a  tube  form  vial  with  a  cork  from  the  inner  sur- 
face of  which  two  small  shallow  holes  have  been  bored,  one  containing  parafor- 
maldehyd,  the  other  camphor.  The  insect  is  mounted  upon  a  pin  stuck  in  the 
cork,  which  latter  is  inserted  and  paraffined  externally. 


C— PREPARATION  OF  NORMAL  SOLUTIONS 


A  normal  solution  is  one  which  contains  the  hydrogen  equivalent  of  an  element, 
expressed  in  grams,  dissolved  in  sufficient  distilled  water  to  make  1000  c.c. 
hydrogen  equivalent  is  the  atomic  weight  of  any  element  divided  by  its  valence.  Ii 
a  base,  salt  or  acid,  we  use  the  molecular  weight  in  grams  divided  by  valence. 


APPENDIX  453 

What  may  be  considered  as  the  valence  of  a  base  is  shown  by  the  number  of 
hydroxyls  combined  with  it;  that  of  an  acid  by  the  number  of  replaceable  hydrogen 
atoms  which  it  contains. 

To  make  a  normal  solution,  dissolve  in  distilled  water  a  weight  in  grams  equal 
to  the  sum  of  the  atomic  weights  of  the  substance  divided  by  its  valence,  and  make 
up  the  volume  to  exactly  1000  c.c. 

NaOH  is  univalent.  Na  =  23.  O  =  i6.  H=i.  Dissolve  40  grams  NaOH  in 
water  and  make  up  to  exactly  1000  c.c. 

Oxalic  acid  is  COOH— COOH+2H2O  which  gives  it  a  molecular  weight  of  126. 
As  it  contains  two  carboxyl  groups  it  is  dibasic,  and  it  is  necessary  to  divide  the 
molecular  weight  by  2,  so  that  for  a  normal  solution  of  oxalic  acid  we  dissolve  63 
grams  in  a  volume  of  distilled  water  made  up  to  1000  c.c. 

If  a  chemical  laboratory  is  not  accessible  one  may  prepare  normal  solutions  with 
an  error  so  slight  as  to  be  unimportant  in  clinical  work  in  the  following  way: 

Sodium  hydrate  being  very  hygroscopic,  it  is  impossible  to  accurately  prepare  a 
normal  solution  by  directly  weighing  out  the  substance.  Instead,  select  perfect 
crystals  of  oxalic  acid,  such  as  can  be  obtained  in  a  drug  store,  and  weigh  out  on  the 
most  accurate  apothecary  scales  obtainable  exactly  6.3  grams  of  the  most  perfect 
crystals  in  the  bottle.  Put  these  preferably  in  a  volumetric  flask  and  make  up  with 
distilled  water  to  1000  c.c.  Less  accurate  is  the  use  of  a  measuring  cylinder.  If 
care  is  used  this  should  give  N/io  solution  of  oxalic  acid  in  which  the  error  is  less 
than  i%. 

Having  N/io  acid  at  hand,  we  may  prepare  N/io  NaOH  in  the  following  way: 
Weigh  out  an  excess  of  sodium  hydrate  (5  grams  of  stick  caustic  soda)  and  dissolve 
in  1 100  c.c.  of  distilled  water.  Take  up  10  c.c.  of  this  solution  with  a  pipette  and  let 
it  run  into  a  beaker.  Add  6  drops  of  phenolphthalein  solution.  This  gives  a  violet- 
pink  color.  Fill  the  burette  with  the  N/io  oxalic  acid  solution  and  let  it  run  into 
the  sodium  hydrate  solution  in  the  beaker  until  the  pink  is  just  discharged.  Reading 
off  the  number  of  cubic  centimeters  of  the  N/io  acid  used,  we  know  the  strength 
of  the  sodium  hydrate  solution.  It  is  well  to  repeat  the  titration  and  take  an 
average. 

If  10.5  c.c.  of  the  oxalic  acid  solution  were  required  it  would  show  that  the  sodium 
hydrate  solution  was  stronger  than  N/io,  as  only  10  c.c.  would  have  been  necessary 
if  the  NaOH  solution  had  been  N/io.  It  is  therefore  necessary  to  dilute  the  sodium- 
hydrate  solution  in  the  proportion  of  10  to  10.5.  Measure  exactly  1000  c.c.  of  the 
too  concentrated  sodium-hydrate  solution  and  add  to  it  50  c.c.  of  distilled  water,  mix 
thoroughly,  and  we  have  1050  c.c.  of  N/io  solution  of  NaOH.  icooX  10.5  =  10,500. 
1 0,500  -1-10  =  1050. 

As  Acidum  hydrochloricum  U.  S.  P.  is  about  two-thirds  water  (68.1%)  to  make 
N/io  HC1,  which  would  require  3.65  in  1000  c.c.,  it  would  be  necessary  to  take  about 
three  times  this  amount  of  U.  S.  P.^cid.  Take  12  c.c.  of  the  acid  and  add  distilled 
water  to  make  1 100  c.c.  Put  10  c.c.  of  this  dilute  solution  in  a  beaker.  Add  phenol- 
phthalein solution  and  titrate.  If  n  c.c.  of  N/io  NaOH  were  required  it  would  be 
necessary  to  add  100  c.c.  of  water  to  a  volume  of  1000  c.c.  of  the  diluted  hydrochloric 
acid.  ioooXii  =  n,ooo-T-io  =  iioo. 

Other  acid  and  alkali  solutions  can  be  made  as  for  N/io  HC1  and  N/io  NaOH. 


454  APPENDIX 

D— CHEMICAL  EXAMINATION  OF  THE  BLOOD 

Blood  Sugar. — Normally  we  have  about  0.1%  sugar,  or  100  mg.  in  100  c.c.  of 
blood.  In  diabetes  we  have  hyperglycaemia  with  an  increase  of  blood  glucose 
to  twice  or  even  eight  times  as  much.  The  determination  of  blood  sugar  is  impor- 
tant in  differentiating  the  so-called  "renal  diabetes"  from  true  diabetes.  In  renal 
diabetes  we  have  a  normal  blood  sugar  content.  Furthermore,  the  sugar  in  the  urine 
rarely  exceeds  i  %  and  this  glycosuria  is  not  affected  by  variations  in  carbohydrate 
intake  as  is  the  case  with  true  diabetes  mellitus.  Furthermore,  there  are  no  symp- 
toms, such  as  thirst,  excessive  polyuria,  loss  of  weight,  etc. 

The  micro-method  of  Bang  is  the  one  used  by  many  workers  in  making  this  deter- 
mination. Objections  to  it  that  we  have  noted  have  been  the  necessity  for  an  accu- 
rate chemical  balance  for  the  weighing  of  the  drop  or  two  of  blood  which  is  taken 
up  on  a  previously  weighed  piece  of  filter-paper.  Again  the  titration  of  the  cuprous 
chloride  with  N/2oo  iodine  solution  has  to  be  carried  out  with  exclusion  of  air  and 
furthermore,  the  previous  boiling  gives  changing  results  according  to  time  of  process. 
In  my  opinion  it  can  only  be  carried  out  accurately  in  the  hands  of  an  experienced 
chemical  worker. 

Myers  and  Fine  Modification  of  Lewis  and  Benedict  Method. — For  taking  blood, 

whether  for  sugar  or  nonprotein  nitrogen  determination,  we  use  the  blood  system 

"described  under  "Blood   Examination."     With  a  graduated  centrifuge   tube  we. 

make  a  blue  pencil  mark  at  2  c.c.  for  sugar  determinations,  or  at  5  c.c.  for  nonprotein 

nitrogen  ones. 

A  small  pinch  of  finely  powdered  potassium  oxalate  is  dusted  into  the  bottom 
of  the  graduated  centrifuge  tube.  As  the  blood  drops  into  the  tube  we  keep  agitating 
the  tube  so  that  the  oxalate  dissolves  and  prevents  coagulation  of  the  blood.  In- 
stead of  the  powdered  potassium  oxalate  we  may  put  i  c.c.  of  a  2%  solution  of  the 
oxalate  in  the  centrifuge  tube  and  make  a  mark  for  the  blood  at  the  3  c.c.  or  6  c.c. 
line.  Folin  uses  a  2  or  5  c.c.  pipette  into  which  potassium  oxalate  has  been  dusted 
and  which  is  connected  with  the  needle  in  the  vein. 

Transfer  the  2  c.c.  of  oxalated  blood,  to  which  has  been  added  exactly  8  c.c.  of 
water,  to  a  test-tube.  Then  add  0.2  gram  of  picric  acid.  Mix  thoroughly  and  after 
standing  five  minutes  filter  through  small  dry  filter.  Three  c.c.  of  filtrate  are  placed 
in  a  tall  test-tube  and  i  c.c.  of  20%  Na2CO3  added.  Tube  is  placed  in  boiling  water 
bath  for  fifteen  to  thirty  minutes.  Now  cool  and  make  volume  up  to  20  c.c.  and 
compare  by  Duboscq  colorimeter  with  a  known  dextrose  solution  (0.1%)  similarly 
treated. 

Instead  of  the  Duboscq  or  Hellige  colorimeter  one  can  use  the  following  method 
which  is  sufficiently  accurate  for  clinical  purposes. 

In  a  case  of  diabetes  make  up  a  0.2%  sugar  solution  instead  of  the  0.1%  and  treat 
as  above  with  the  picric  acid.  Then  make  up  accurately  to  20  c.c.  in  a  graduated 
cylinder.  Now  with  the  20  c.c.  of  yellow-colored  solution  from  the  blood  in  a 
tube  between  the  thumb  and  forefinger  of  the  left  hand  we  add  to  the  empty 
tube  alongside  the  known  0.2%  solution  from  the  graduated  cylinder.  When  the 
colors  match  we  read  off  the  amount  used  from  tfce  cylinder,  and  calculate  the 
strength  of  the  blood  sugar  tube.  If  i$  c.c.  were  required  it  would  show  that  the 
sugar  of  the  blood  equalled  0.15%. 


APPENDIX 


455 


Nonprotein  Nitrogen  of  Blood. — Normally  we  have  from  25  to  35  mg.  of  non- 
protein  N  in  100  c.c.  of  blood.  This  is  greatly  increased  in  chronic  nephritis  and 
especially  in  uraemia.  The  nonprotein  N  is  more  easily  and  accurately  determined 
than  the  urea  N.  Of  course,  the  urea  N  makes  up  about  one-half  of  the  normal 
nonprotein  N  and  in  uraemic  conditions  it  may  constitute  from  80  to  90%  of  the 
nonprotein  N.  Hawk  gives  urea  N  as  12  to  23  mg.  per  100  c.c.  of  blood.  In 
uraemia  it  may  amount  to  70  to  300  mg.  with  a  nonprotein  N  increase  of  from  90 
to  350  mg.  per  100  c.c.  of  blood. 

To  Estimate  Nonprotein  N. — Take  5  c.c.  blood  in  a  graduated  centrifuge  tube 
with  powdered  potassium  oxalate  as  for  blood  sugar.  Be  sure  not  to  use  ammonium 
oxalate.  Now  gradually  add,  with  constant  stirring,  75  c.c.  of  absolute  alcohol, 
mix  thoroughly,  filter  through  dry  filter,  wash  precipitate  with  several  small  portions 
of  alcohol,  evaporate  filtrate  and  washings  to  dryness  on  water-bath,  using  a  small 
porcelain  dish  for  the  purpose,  and  with  smallest  possible  quantity  of  water  take  up 
and  transfer  residue  to  a  small  flask.  Add  i  to  2  grams  potassium  sulphate  and  2 
c.c.  of  concentrated  sulphuric  acid,  place  over  a  small  flame  and  heat  until  all  water 
is  driven  off  and  the  contents  of  flask,  after  having  become  thoroughly  charred, 
have  again  become  a  pale  yellow  or  colorless.  Cool,  add  about  20  c.c.  of  water, 
again  cool,  add  4  or  5  drops  of  phenolphthalein,  gradually  add  10%  solution  of  sod- 
ium hydrate,  keeping  contents  of  flask  cool  by  frequent  immersion  in  cold  water, 
until  exact  point  of  neutrality  is  reached.  Now  add  5  grams  of  neutral  potassium 
oxalate,  stir  until  dissolved  and  then  add  about  10  c.c.  of  neutral  20%  formaldehyde 
(50%  formalin)  and  then  from  burette  add  N/50  NaOH  until  permanent  pink 
color  is  obtained. 

The  number  of  cubic  centimeters  N/50  NaOH  required  times  0.28  equals  number 
milligrams  nonprotein  nitrogen  in  5  c.c.  of  blood. 

Urea  N  of  Blood. — The  following  method  suggested  by  Dunning  has  been 
modified  in  our  laboratory.  For  urea  estimation  take  5  c.c.  blood  as  for  nonprotein 
N  and  dilute  with  25  c.c.  distilled  water  and  add  30  grams  of  crystallized  sodium 
sulphate  or  13  grams  of  anhydrous  sodium  sulphate.  The  mixture  is  then  warmed 
to  35°C.  when  the  sodium  sulphate  is  dissolved  and  the  blood  proteins  precipitated. 
The  mixture  is  then  made  up  to  50  c.c.  with  a  saturated  solution  of  sodium  sulphate, 
thoroughly  shaken  and  allowed  to  stand  a  few  minutes.  It  is  then  filtered  and 
25  c.c.  of  the  filtrate  used  for  the  test  and  10  c.c.  for  a  control.  Each  ic.c.c.  repre- 
sents i  c.c.  of  the  oxalated  blood. 

To  25  c.c.  of  the  nitrate  add  distilled  water  to  100  c.c.  and  one  finely  powdered 
Urease-Dunning  tablet  and  digest  for  i  hour  at  50°  C.  or  over  night  at  room  tem- 
perature. 

After  digestion  filter  through  double  dry  filter  and  to  40  c.c.  of  this  nitrate,  which 
represents  i  c.c.  of  the  blood,  add  2  drops  of  methyl  orange. 

Now  add  2  drops  of  methyl  orange  to  the  control  and  titrate  with  N/2O  HC1 
to  a  faint  pink  color.  Then  titrate  the  urease-treated  solution  to  exactly  the  same 
shade  of  pink  as  the  control. 

The  amount  of  N/20  HC1  required  to  neutralize  the  urease-treated  solution,  less 
the  amount  required  for  the  control,  multiplied  by  0.0015  will  give  the  urea  in  i  c.c. 
of  the  blood:  or  multiplied  by  0.0007  will  give  the  urea  nitrogen  in  the  i  c.c.  of  blood. 

The  end  reaction  is  not  at  all  sharp  and  requires  considerable  experience  to  deter- 


456  APPENDIX 

mine  it.     The  control  should  be  titrated  before  the  test  solution  acted  upon  by  the 
urease  and  the  shade  of  the  test  solution  should  correspond  to  the  control.     About 

2  drops  of  a  0.05%  aqueous  solution  of  methyl  orange  should  be  used  for  50  c.c.  of 
test  solution. 

Hydrogen-ion  Concentration  of  the  Blood. — Levy,  Rowntree  and  Marriott  have 
proposed  this  determination  as  an  index  of  the  reaction  of  the  blood.  The  test 
may  be  made  either  on  oxalated  blood  or  with  serum.  The  hydrogen-ion  concen- 
tration of  the  serum  of  normal  persons  varies  from  p  H  7.6  to  p  H  7.8,  that  of  oxa- 
lated blood  from  p  H  7.4  to  p  H  7.6.  The  exact  point  of  neutrality,  p  H  7,  is  only 
reached  in  severe  uncompensated  acidosis  and  a  reaction  of  p  H  8  is  only  possibly 
obtainable  after  administration  of  alkalis. 

One  to  3  c.c.  of  clear  serum  or  of  blood  is  run,  by  means  of  a  blunt-pointed 
pipette,  into  a  dialyzing  sac  which  has  been  washed  inside  and  outside  with  salt 
solution  and  which  has  been  tested  for  leaks  by  filling  with  the  salt  solution.  The 
sac  is  lowered  into  a  small  test-tube  (100  x  10  mm.,  inside  measurements)  containing 

3  c.c.  of  the  salt  solution,  until  the  fluid  on  the  outside  of  the  sac  is  as  high  as  on  the 
inside.     From  five  to  ten  minutes  are  allowed  for  dialysis.     The  collodion  sac  is 
removed  and  five  drops  of  the  indicator  (an  aqueous  0.01%  solution  of  phenol- 
sulphonphthalin)  are  thoroughly  mixed  with  the  dialysate.     The  tube  is  then  com- 
pared with  the  series  of  standards  until  the  corresponding  color  is  found,  which 
indicates  the  hydrogen-ion  concentration  present  in  the   dialysate. 

These  tests  may  be  carried  out  with  3  c.c.  of  blood  or  serum.  The  same  results 
are  obtained  with  i  c.c.  of  blood  or  serum  on  the  inside  of  the  sac,  and  with  this 
amount  it  is  immaterial  whether  there  is  i  or  3  c.c.  of  salt  solution  on  the  outside. 

For  the  comparison  of  tubes  with  standards  a  good  light  (natural  or  artificial) 
and  a  white  background  are  requisites.  Readings  must  be  made  immediately. 
The  tube  matching  most  closely  is  selected  and  also  the  tubes  on  either  side  of  it. 
These  are  critically  inspected  against  a  white  background.  Changing  the  order 
of  the  tubes  often  makes  differences  more  apparent. 

Hynson,  Westcott  and  Co.  prepare  a  set  of  standards  with  the  h  drogen-ion  con- 
centration marked  on  each  ampule.  The  dialyzing  collodion  sacs  are  made  as  for 
the  Abderhalden  test  or  they  may  be  purchased. 


E— CHEMICAL  EXAMINATION  OF  URINE 


For  the  prevention  of  decomposition  when  a  urine  is  not  examined  shortly  after 
voiding,  chloroform  (10  to  20  drops  added  to  a  tightly  corked  bottle)  or  formalin 
(4  or  5  drops  to  a  pint  of  urine)  are  ordinarily  employed.  Formalin  is  better  for 
microscopical  material,  but,  owing  to  its  reducing  power,  should  be  substituted  by 
boric  acid  in  urine  to  be  examined  for  sugar.  For  clearing  urine,  turbid  by  reason 
of  bacteria,  rubbing  up  with  Talcum  purificat.,  U.  S.  P.,  and  filtering  is  recom- 
mended. //  is  advisable  to  call  for  a  fresh  specimen  and  not  to  examine  a  decomposed 
urine  either  for  casts  or  albumin, 

A  twenty-four-hour  specimen  is  necessary  for  accurate  work.  The  urine  should 
be  collected  in  clean  separate  bottles.  Where  pus  comes  from  the  bladder  the 
proportion  of  pus  in  each  bottle  will  be  practically  the  same;  if  from  the  kidneys 
the  amount  will  vary  in  the  different  bottles. 


ter 


APPENDIX  457 

The  amount  of  urine  varies  in  different  individuals  (water  or  beer  habit).  It  is 
usually  given  as  from  1000  to  1500  c.c. 

Long  proposes  to  substitute  2.6  for  Haeser's  coefficient  which,  if  multiplied  by 
the  two  final  figures  of  the  specific  gravity  taken  at  25°C.,  gives  the  weight  of  urin- 
ary solids  in  1000  c.c. 

Albumin. — Practically  serum  albumin  alone  is  clinically  important. 

The  two  usual  tests  are  i.  Heat  test  and  2.  Heller's  nitric  acid  test.  For  the 
former,  add  3  to  10  drops  of  5%  acetic  acid  to  the  perfectly  clear  urine  in  a  test- 
tube  and  bring  to  a  boil.  By  boiling  the  upper  portion  a  turbidity  in  contrast  with 
the  clear  lower  portion  may  be  obtained.  I  usually  prefer  to  heat  the  urine  and  after 
the  boiling  add  the  5%  acetic  acid,  drop  by  drop.  This  will  clear  up  the  turbidity 
due  to  phosphates. 

A  more  delicate  test  for  albumin  is  the  following:  Add  to  a  test-tube  half  filled 
with  filtered  urine  one-fifth  its  volume  of  a  saturated  aqueous  solution  of  sodium 
chloride;  heat  to  the  boiling-point;  add  2  to  5  drops  of  50%  acetic  acid  and 
heat  again.  This  test  may  serve  to  distinguish  nucleo-albumin,  as  most  forms  of 
nucleo-proteid  found  in  urine  do  not  react  to  the  test,  while  serum  albumin  does. 
Thus  where  a  positive  nitric  acid  test  is  present,  and  no  precipitate  occurs  with 
this  test,  the  proteid  present  is  usually  nucleo-proteid. 

For  Heller's  test,  pour  a  small  amount  of  nitric  acid  into  a  narrow  test-tube 
and,  while  holding  the  tube  at  an  angle  of  about  45  degrees,  superimpose  a  layer  of 
the  urine  to  be  tested,  which  is  delivered  drop  by  drop  from  a  pipette  and  allowed 
to  flow  down  the  side  of  the  tube. 

This  test  can  be  converted  into  a  quantitative  one  which  is  sufficiently  accurate 
for  clinical  purposes.  It  is  based  on  the  fact  that  a  specimen  of  urine  containing 
0.003  %  of  albumin  will  give  a  perceptible  ring  at  the  layering  of  the  urine  and  acid 
in  two  minutes.  If  the  ring  appears  at  once  or  in  a  few  seconds"  the  albumin  con- 
tent is  greater.  From  the  qualitative  test  an  idea  can  be  formed  as  to  the  amount 
of  albumin  which  the  urine  contains,  a  heavy  ring  forming  immediately  showing  a 
considerable  albumin  content.  Probably  the  highest  elimination  of  albumin  is 
found  in  chronic  parenchymatous  nephritis  where  it  may  run  from  i  to  3%.  In 
an  ordinary  case  of  acute  nephritis  0.5%  would  be  an  average  content. 

Recently  I  have  been  using  for  both  qualitative  and  quantitative  albumin  tests 
the  apparatus  shown  in  Fig.  7.  This  is  simply  a  5-inch  piece  of  ^-inch  soft 
glass  tubing  heated  at  a  point  2  inches  from  one  end,  drawn  out  about  2  inches 
and  bent  to  form  a  U  tube  with  one  end  shorter  than  the  other.  This  form  of 
tube  enables  one  to  perform  two  tests  with  the  same  column  of  nitric  acid  and  is 
easily  cleaned  and  dried.  They  may  be  kept  suspended  around  a  glass  tumbler's 
rim.  Taking  up  a  small  amount  of  nitric  acid  with  a  capillary  bulb  pipette  it  is 
deposited  in  the  capillary  curve  of  the  bent  tube.  This  acid  pipette  should  be 
kept  attached  to  the  acid  bottle.  With  a  second  pipette  the  urine  is  deposited  in 
the  short  arm  of  the  U  tube  and  the  presence  of  albumin  shows  by  a  distinct  ring 
at  the  junction  of  urine  and  acid  in  the  clear  capillary  tubing.  The  long  arm  will 
serve  for  the  introduction  of  a  different  specimen  of  urine  for  the  albumin  test. 

For  quantitative  test  we  dilute  the  filtered  urine  with  one  or  more  parts  of  nor- 
mal salt  solution  according  to  the  intensity  of  the  albumin  ring.  A  very  convenient 
way  of  making  the  dilution  is  with  a  graduated  centrifuge  tube.  Make  a  i  to 


458  APPENDIX 


t  in  the 


10  dilution  of  the  urine,  mix  and  draw  up  with  a  bulb  pipette  and  deposit  i 
short  arm  of  the  U  tube  containing  nitric  acid.  A  distinct  ring  forms  in  two  to 
three  seconds.  Pour  off  one-half  of  the  diluted  urine  and  make  up  with  an  equal 
amount  of  saline.  Deposit  this  i  to  20  dilution  in  the  long  arm.  The  ring  forms 
in  about  a  minute.  With  further  testing  it  is  found  that  a  i  to  40  dilution  shows  a 
perceptible  ring  in  just  two  minutes.  This  final  and  successful  dilution  multiplied 
by  0.0033  gives  the  percentage  of  albumin  in  the  urine  (40  X  0.0033  =  0.13%). 

Should  it  be  desired  to  determine  the  nature  of  the  proteids  present  either  in 
urine  or  in  exudates  or  transudates  the  following  method  is  applicable.  Determine 
the  percentage  of  total  proteid  by  the  method  employed  above.  Then  throw  down 
the  globulins  by  the  addition  of  an  equal  amount  of  a  saturated  solution  of  ammo- 
nium sulphate,  filter  and  estimate  the  proteid  content  of  the  filtrate.  The  differ- 
ence between  that  and  the  total  gives  the  percentage  of  globulin.  The  filtrate  is 
now  treated  with  5%  acetic  acid  until  a  precipitate  of  nucleo-proteid  ceases  to  form; 
the  fluid  is  filtered  and  the  clear  filtrate  (which  should  not  show  any  turbidity  with 
a  drop  of  5%  acetic  acid)  is  tested  for  its  proteid  content,  which  represents  the 
serum  albumin.  When  the  combined  percentage  of  globulins  and  serum  albumin 
is  subtracted  from  the  total  proteid  percentage  we  have  the  percentage  of  nucleo- 
proteid. 

Esbach's  Quantitative  Method  for  Albumin. — The  use  of  the  Esbach  tube  is  at- 
tended with  some  uncertainty,  whether  using  the  original  Esbach  solution  or  that 
devised  by  Tsuchiya,  the  precipitate  at  times  refusing  to  settle.  The  method  of 
using  the  tube  is  to  add  the  urine  to  the  U  mark  and  then  the  reagent  to  the  R  mark, 
mix,  and  allow  to  stand  in  upright  position.  If  Esbach's  reagent  has  been  used  the 
reading  is  made  at  the  end  of  twenty-four  hours,  but  when  Tsuchiya's  reagent  is 
employed  the  reading  is  made  at  the  end  of  two  hours.  The  number  at  the  end  of 
the  line  which  corresponds  to  the  upper  limit  of  precipitate  will  be  the  number 
of  grams  of  dry  albumin  per  liter  of  urine.  Esbach's  reagent  consists  of  10  grams 
of  picric  and  20  grams  of  citric  acid  dissolved  in  i  liter  of  water.  Tsuchiyas' 
reagent  is  made  by  dissolving  1.5  grams  of  phosphotungstic  acid  in  a  mixture  of  5 
c.c.  strong  hydrochloric  acid  and  95  c.c.  of  95%  alcohol.  In  this  method  also 
dilution  must  be  resorted  to  if  albumin  or  specific  gravity  are  excessive. 

Nucleo -protein. — Increased  quantities  of  this  protein  occur  in  pyelitis,  nephritis, 
and  inflammations  of  the  bladder. 

For  its  detection  albumin,  if  present  in  any  considerable  quantity,  must  be  re- 
moved by  boiling  as  described  above.  Then  place  10  c.c.  of  this  urine  in  a  small 
beaker,  dilute  with  3  volumes  of  water  and  make  the  reaction  very  strongly 
acid  with  acetic  acid.  If  the  solution  becomes  turbid  it  is  an  indication  that 
nucleo-protein  is  present. 

Bence-Jones  Body. — (Albumose.)  Perform  the  heat  test  for  albumin.  The 
appearance  of  a  heavy  precipitate  which  partially  clears  on  boiling  suggests  albu- 
mose.  If  albumose  is  present  a  cloud  will  appear  in  the  filtrate  on  cooling.  The 
precipitate  formed  with  nitric  acid,  if  due  to  albumose,  disappears  with  heat,  that 
of  serum  albumin  does  not. 

As  another  test  for  the  Bence-Jones  body,  usually  present  in  multiple  myelomata, 
that  of  Boston  is  of  value.  Mix  15  c.c.  urine  in  a  test-tube  with  an  equal  amount  of 
saturated  NaCl  solution.  Add  2  c.c.  of  40%  NaOH  solution  and  shake  the  contents 


APPENDIX  459 

of  the  tube  thoroughly.  Heat  the  upper  contents  of  the  tube  to  boiling  and  add  lead 
acetate  solution  (10%)  drop  by  drop  continuing  the  heating..  A  brown  to  black 
precipitate  (sulphur)  shows  this  form  of  albumin. 

In  tests  requiring  the  removal  of  albumin  boil 'the  urine  and  add  dilute  acetic 
acid  until  the  precipitate  is  flocculent,  then  filter. 


SUGAR 

Fehling. — Pour  equal  parts  of  Fehling's  copper  solution  (34.639  grams  of  copper 
sulphate  in  500  c.c.  of  water)  and  Fehling's  alkali  solution  (173  grams  sodium  potas- 
sium tartrate  and  50  grams  sodium  hydrate  in  500  c.c.  water)  into  a  test-tube.  Mix 
and  dilute  the  deep  blue  solution  with  2  parts  of  water.  Heat  the  upper  portion 
of  the  diluted  Fehling's  solution  in  the  flame  to  boiling  and  drop  in  from  a  pipette 
the  urine  to  be  examined.  A  yellowish  to  red  precipitate  shows  the  presence  of 
sugar. 

Fehling's  test  will  show  the  presence  of  J^oo  of  i%  of  glucose  in  an  aqueous 
solution  but  is  vastly  less  delicate  for  sugar  in  urine.  This  is  due  to  the  power 
of  the  creatinin  in  urine  of  holding  the  reduced  suboxide  of  copper  in  solution. 
An  important  point  is  that  the  creatinin  is  broken  up  by  prolonged  boiling  hence 
the  puzzling  precipitates  one  gets  at  times  after  a  long  period  of  boiling  are  explained 
in  this  way.  Glycuronic  acid  may  cause  a  doubtful  reaction.  If  the  precipitated 
cuprous  oxide  is  in  very  fine  granules  the  color  is  greenish,  if  less  fine,  greenish 
yellow  and  if  quite  coarse,  reddish. 

Creatinin  holds  in  solution  the  copper  suboxide  formed  by  uric  acid  as  well  as 
that  resulting  from  very  small  glucose  content  of  urine. 

As  a  test  for  doubtful  glycosuria  it  is  well  to  give  100  grams  of  pure  glucose.  A 
normal  person  should  deal  with  such  an  amount  without  showing  sugar  reaction  of 
the  urine. 

Phenylhydrazin  (Kowarsky). — Mix  5  drops  of  pure  phenylhydrazin  in  a  test- 
tube  with  10  drops  of  glacial  acetic  acid.  Shake  lightly  and  add  15  drops  of  satu- 
rated solution  of  NaCl.  This  makes  a  pasty  mixture.  Now  add  10  c.c.  of  the  urine 
and  bring  carefully  to  a  boil  over  a  small  flame  and  continue  to  boil  gently  for  two 
minutes.  Upon  cooling  a  yellowish  crystalline  precipitate  falls  more  or  less  rapidly 
according  to  the  sugar  content  of  the  urine.  If  the  urine  contains  0.2%  or  more  of 
sugar  the  precipitate  appears  in  a  few  minutes.  The  test  is  sensitive  for  0.03%  of 
sugar. 

Fermentation  Test.— This  is  the  surest  test  for  sugar  in  the  urine.  It  will  show 
the  presence  of  0.05%  of  glucose.  Instead  of  the  Einhorn  apparatus  one  may  be 
extemporized  by  taking  a  50  c.c.  cylinder,  filling  it  to  overflowing  with  the  urine 
which  has  previously  been  rubbed  up  with  a  piece  of  compressed  yeast  the  size  of  a 
hazel  nut.  The  urine-should  be  made  acid  with  tartaric  acid  to  prevent  ammoniacal 
decomposition  with  the  formation  of  CO2.  A  small  3-inch  test-tube  is  filled  with  the 
yeast-treated  urine  and  dropped  mouth  downward  into  the  50  c.c.  cylinder.  The 
apparatus  is  incubated  for  twenty-four  hours  and  the  presence  of  gas  in  the  closed 
end  of  the  test-tube  shows  that  sugar  was  present.  A  control  to  determine  that  the 
yeast  does  not  contain  sugar  is  advisable.  To  utilize  this  test  as  a  quantitive  one, 
first  accurately  take  the  specific  gravity  of  the  urine;  then  add  the  yeast  and  fill 


460  APPENDIX 

the  test-tube  and  cylinder  as  directed  above.  Next  pour  off  or  pipette 
urine  exactly  to  the  50  c.c.  mark.  Incubate  for  twenty-four  to  forty-eight  hours 
and  make  up  the  loss  by  evaporation,  with  distilled  water.  After  the  urine  has 
cooled  down  to  room  temperature  the  contents  of  tube  and  cylinder  are  thoroughly 
mixed  (the  small  tube  having  been  withdrawn  with  a  pair  of  forceps),  then  filtered 
to  remove  the  sediment  of  yeast  and  then  brought  to  the  exact  original  volume  of 
50  c.c.  with  distilled  water  to  make  up  the  loss  by  evaporation.  (If  there  should  be 
doubt  as  to  the  completion  of  the  fermentation  of  the  glucose  a  qualitative  test  for 
sugar  can  be  made.)  The  specific  gravity  is  again  taken  and  the  difference  between 
this  and  the  first  reading  multiplied  by  0.23.  Example:  Specific  gravity  of  unfer- 
mented  urine,  1.030,  that  of  urine  after  incubation,  1.022.  Difference,  8X0.23  = 
1.84%. 

It  is  advisable  to  have  two  good  urino meters,  one  to  register  from  1000  to  1025, 
a  second  to  register  from  1025  to  1050. 

Benedict's  New  Method  for  Quantitative  Determination  of  Sugar  in  Urine. 

The  solution  for  quantitative  work  has  the  following  composition:  . 

Copper  sulphate  (pure  crystallized) 18  .o  gm. 

Sodium  carbonate — crystallized  (100  grams  of  anhydrous  salt 

will  answer) 200 .  o  gm. 

Sodium  or  potassium  citrate 200.0  gm. 

Potassium  sulphocyanate 125 .o  gm. 

5%  potassium  ferrocyanid  solution 5.0  c.c. 

Distilled  water  to  make  total  volume  of 1000 .  o  c.c. 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate  and  sulphocyanate  in  enough 
water  to  make  about  800  c.c.  of  the  mixture,  and  filter  if  necessary.  Dissolve  the 
copper  sulphate  separately  in  about  100  c.c.  of  water  and  pour  the  solution  slowly 
into  the  other  liquid,  with  constant  stirring.  Add  the  ferrocyanid  solution,  cool  and 
dilute  to  exactly  i  liter.  Of  the  various  constituents,  the  copper  salt  only  need  be 
weighed  with  exactness.  Twenty-five  c.c.  of  the  reagent  are  reduced  by  50  mg.  of 
glucose. 

Sugar  estimations  are  conducted  as  follows:  The  urine,  10  c.c.  of  which  should 
be  diluted  with  water  to  100  c.c.  (unless  the  sugar  content  is  believed  to  be  low),  is 
poured  into  a  50  c.c.  burette  up  to  the  zero  mark.  Twenty-five  c.c.  of  the  reagent 
are  measured  with  a  pipette  into  a  porcelain  evaporating  dish  (25-30  cm.  in  diameter), 
10  to  20  grams  of  crystallized  sodium  carbonate  (or  one-half  the  weight  of  the  anhy- 
drous salt)  are  added,  together  with  a  small  quantity  of  powdered  pumice-stone  or 
talcum,  and  the  mixture  heated  to  boiling  over  a  free  flame  until  the  carbonate  has 
entirely  dissolved.  The  diluted  urine  is  now  run  in  from  the  burette,  rather  rapidly 
until  a  chalk  white  precipitate  forms,  and  the  blue  color  of  the  mixture  begins  to 
lessen  perceptibly,  after  which  the  solution  from  the  burette  must  be  run  in  a  few 
drops  at  a  time,  until  the  disappearance  of  the  last  trace  of  blue  color,  which  marks 
the  end  point.  The  solution  must  be  kept  vigorously  boiling  throughout  the  entire 
titration.  If  the  mixture  becomes  too  concentrated  during  the  process,  water  may 
be  added  from  time  to  time  to  replace  the  volume  lost  by  evaporation.  The  cal- 


APPENDIX  461 

culation  of  the  percentage  of  sugar  in  the  original  sample  of  urine  is  very  simple. 
The  25  c.c.  of  copper  solution  are  reduced  by  exactly  50  mg.  of  glucose.     Therefore 
the  volume  run  out  of  the  burette  to  effect  the  reduction  contained  50  mg.  of  the 
sugar.     When  the  urine  is  diluted  i  :io,  as  in  the  usual  titration  of  diabetic  urines, 
the  formula  for  calculating  the  per  cent,  of  sugar  is  the  following: 
0.050 
•.£--  times  1000  equals  per  cent,  in  original  sample,wherein  X  is  the  number  of 

cubic  centimeters  of  the  diluted  urine  required  to  reduce  25  c.c.  of  the  copper 
solution. 

In  the  use  of  this  method  chloroform  must  not  be  present  during  the  titration. 
If  used  as  a  preservative  in  the  urine  it  may  be  removed  by  boiling  a  sample  for  a 
few  minutes,  and  then  diluting  to  its  original  volume. 

This  solution  will  keep  indefinitely  and  it  is  claimed  by  Benedict,  that  compari- 
son with  the  polariscope  and  by  Allihn's  gravimetric  process  will  show  it  to  be  more 
accurate  than  any  of  the  ordinarily  used  methods. 

APPROXIMATE  QUANTITATIVE  ESTIMATION  with  Fehling's  solution.  (One  c.c.  of 
Fehling's  solution  is  reduced  by  5  mg.  glucose.) 

Measure  off  2  c.c.  of  Fehling's  solution  in  a  pipette  and  put  in  a  test-tube  or 
small  beaker  and  dilute  with  20  c.c.  of  water. 

Bring  the  diluted  Fehling's  to  boiling  and  drop  in  drop  by  drop  the  urine  from 
a  dropping-bottle  for  which  the  number  of  drops  per  cubic  centimeter  has  been 
noted.  Estimating  20  drops  to  the  cubic  centimeter  if  2  drops  of  urine  are  re- 
quired to  reduce  the  copper  it  would  show  a  sugar  percentage  of  the  urine  of  10. 
Four  drops  5%,  8  drops  2.5%,  16  drops  1.25%,  32  drops  0.6%,  64  drops  0.3%,  100 
drops  0.2%. 

Nylander's  Bismuth  Reduction  Test. — Put  5  c.c.  of  urine  in  a  test  tube  and  add 
0.5  c.c.  Nylander's  reagent,  then  heat  for  five  minutes  in  a  boiling  water  bath.  If 
sugar  be  present  the  mixture  will  darken  to  become  black  on  standing.  For  the 
reagent  digest  2  grams  of  bismuth  subnitrate  and  4  grams  Rochelle  salts  in  100  c.c. 
of  10%  KOH  solution.  Cool  and  filter. 

Urinary  Tests  in  Connection  with  Acidosis 

The  determination  of  the  ammonia  quotient,  which  is  the  ratio  of  N  eliminated 
as  ammonia  to  total  nitrogen  elimination,  has  assumed  great  importance  by  reason 
of  its  connection  with  various  forms  of  acid  intoxication,  as  in  diabetes,  pernicious 
vomiting  of  pregnancy,  and  various  hepatic  diseases. 

The  degree  of  acidosis  is  better  determined  by  the  quantitative  estimation  of 
nitrogen  elimination  as  ammonia  than  by  estimating  quantitatively  the  amount  of 
diacetic  and  /3-oxybutric  acid  in  the  urine.  Normally  we  have  about  0.7  gram  of 
ammonia  eliminated  daily.  In  acidosis  this  may  rise  to  5  or  10  grams  and  instead 
of  being  from  3  to  5%  of  the  total  N,  it  may  amount  to  30  to  50%. 

In  the  acidosis  connected  with  chronic  nephritis  it  has  been  found  that  the  pre- 
formed ammonia  is  often  below  normal,  indicating  a  defect  in  the  normal  neutraliz- 
ing action  of  ammonia.  Where  there  is  excessive  formation  of  acid  bodies  as  in 
diabetes,  or  when  liver  cell  degeneration  interferes  with  the  normal  conversion  of 
ammonia  into  urea  we  have  an  increase  in  urinary  ammonia  output. 


462  APPENDIX 

Reaction. — Urine  may  be  quite  acid  in  acidosis.  The  reaction  of  the  mixed 
twenty-four-hour  excretions  is  acid  to  litmus.  At  times  during  this  period,  especi- 
ally after  meals,  the  reaction  may  be  alkaline.  It  is  not  positively  known  to 
what  the  acidity  is  due,  some  authorities  considering  it  due  to  sodium  dihydro- 
gen  phosphate,  while  others  attribute  it  to  organic  acids.  The  acidity  being  very 
largely  due  to  food,  naturally  it  would  vary  in  health,  as  the  nature  of  food  varies. 
The  degree  of  acidity  under  normal  conditions  is  such  that  from  40  to  60  c.c.  of 
N/i  alkali  will  be  required  to  neutralize  the  twenty-four-hour  excretion. 

The  acidity  can  be  determined  by  measuring  25  c.c.  of  sample  into  beaker,  adding 
75  c.c.  of  distilled  water,  and  also,  if  available,  10  grams  of  neutral  potassium  oxalate 
and  finally  3  or  4  drops  of  phenolphthalein  From  a  burette  add  N/io  sodium 
hydrate  until  a  permanent  faint  red  or  pink  color  is  obtained.  High-colored  urines 
must  be  diluted  with  more  than  75  c.c.  of  water  before  the  end  reaction  can 
readily  be  detected.  Normally,  6  to  8  c.c.  neutralizes  25  c.c.  urine. 

Formalin  Method  for  the  Estimation  of  Ammonia 

Free  ammonia  reacts  with  formalin  to  form  hexamethylenetetramine.  If  sodium 
hydrate  is  added  to  neutralized  urine  in  the  presence  of  formalin  free  ammonia 
is  liberated  and  reacts  with  the  formalin.  So  soon  as  all  the  ammonia  has  been 
liberated,  the  end  reaction  occurs. 

Ronchese  first  utilized  this  principle  and  Mathison  found  that  potassium  oxalate 
made  the  end  reaction  sharper.  Brown  found  that  preliminary  clearing  with 
lead  subacetate  made  the  end  reaction  still  sharper  and  removed  certain  nitrogenous 
substances  which  reacted  with  formalin  making  the  result  only  about  5%  higher 
than  with  Schaffer's  method.  The  technic  is  as  follows:  About  60  c.c.  of  filtered 
urine  are  treated  with  3  grams  of  basic  lead  acetate,  well  stirred,  allowed  to  stand 
a  few  minutes  and  filtered.  The  filtrate  is  treated  with  2  grams  of  neutral  potas- 
sium oxalate  well  stirred  and  filtered;  10  c.c.  of  the  clear  filtrate  are  diluted  to  50 
c.c.  with  distilled  water;  a  few  drops  of  i%  phenolphthalein  solution  are  added. 
The  mixture  will  be  slightly  alkaline  or  acid.  Five  grams  potassium  oxalate 
are  added  and  stirred.  It  is  exactly  neutralized  with  decinormal  NaOH  or  H2SO4. 
Twenty  c.c.  of  20%  conmercial  formalin,  previously  made  neutral,  are  added,  and 
the  solution  again  titrated  with  decinormal  NaOH  to  neutralization.  Every  cubic 
centimeter  of  decinormal  NaOH  corresponds  to  0.0017  gram  NH8.  The  quantity 
of  ammonia  is  then  calculated  on  the  basis  of  the  twenty-four-hour  volume. 
Example:  The  10  c.c.  of  urine  required  4  c.c.  N/io  NaOH  to  give  a  pink  color. 
4X0.0017  =  0.0068.  Then  100  c.c.  urine  would  contain  0.068  and  1000  c.c. 
(twenty-four-hour  urine  amount)  0.68  gram  of  ammonia. 

ESTIMATION  OF  TOTAL  NITROGEN 

Principle. — The  nitrogenous  material  of  the  urine  is  converted  into  ammonium 
sulphate  on  boiling  with  H2SO<.  The  ammonia  is  then  estimated  as  described 
under  estimation  of  ammonia  by  the  formalin  method. 

Technic. — Solutions  required: 

i.  Twenty  per  cent,  commercial  formalin  previously  made  neutral  with  NaOH. 


APPENDIX 


463 


2.  N/io  NaOH. 

3.  Forty  per  cent.  NaOH. 

Ten  c.c.  of  filtered  urine  are  pipetted  into  a  Kjeldahl  or  Koch  flask;  10  c.c.  of 
concentrated  H2SO4  and  10  grams  K2SO4  are  added.  The  mixture  is  heated  over 
a  free  flame,  gently  at  first  to  avoid  foaming,  and  is  finally  brought  to  a  boil,  which 
is  continued  until  the  mixture  is  perfectly  clear,  usually  requiring  forty-five  minutes 
to  an  hour.  The  contents  are  cooled  and  quantitatively  transferred  to  a  200  c.c. 
volumetric  flask  and  i  c.c.  of  phenolphthalein 
solution  added.  The  greater  part  of  the 
acidity  is  now  neutralized  by  adding  about 
30  c.c.  of  the  40%  NaOH.  It  is  cooled 
under  a  water  tap  and  made  up  to  the  200  c.c. 
mark;  10  c.c.  are  taken,  diluted  in  50  c.c.  with 
distilled  water  and  exactly  neutralized  with 
N/io  NaOH.  Twenty  c.c.  of  the  formalin 
solution  are  now  added  and  the  titration 
again  performed.  The  pink  end  reaction  is 
beautifully  clear  and  sharp.  The  second 
reading  multiplied  by  the  factor  0.0014  gives 
the  amount  of  nitrogen  in  grams  in  10  c.c. 
of  the  fluid.  It  is  tthen  computed  for  the 
twenty-four-hour  volume  as  for  N,  eliminated 
as  ammonia.  Example:  It  required  5  c.c. 
N/io  NaOH — 5X0.0014  =  0.007.  As  origi- 
nal 10  c.c.  were  diluted  to  200,  the  10  c.c. 
taken  for  titration  would  onlybe^o;  hence 
0.007X20  =  0.14  gram  for  10  c.c.  or  1.4  for 
100  c.c.  or.  14  grams  for  1000  c.c. 

UREA  ESTIMATIONS 

The  amount  of  urea,  which  represents  from 
85  to  90%  of  the  total  nitrogen,  is  usually 
determined  instead  of  the  total  N.  The 
hypobromite  and  hypochlorite  methods  are, 
however,  lacking  in  accuracy,  and  more  exact 
methods  of  urea  estimation  are  more  time-con- 
suming than  the  one  just  given  for  total  N. 

Probably  the  most  convenient  test  for  urea  is  the  hypobromite  method,  using 
the  Doremus  ureometer  with  a  side  tube  connected  to  the  closed  arm  of  the  fermenta- 
tion tube  by  a  glass  stop  cock. 

The  reagent  is  prepared  by  taking  70  c.c.  of  a  30%  stock  solution  of  NaOH, 
diluting  it  with  180  c.c.  water  and  then  adding  5  c.c.  of  bromine,  stirring  until  the 
bromine  is  dissolved.  This  solution  if  stored  in  a*cool  dark  place  will  keep  about 
one  week. 

The  urine  to  be  tested  must  be  free  from  sugar  and  albumin  and  contain  less  than 
i%  of  urea.  Ordinarily  the  urine  must  be  diluted  two  to  four  times  to  obtain  a 
specimen  containing  less  than  i%.  In  using  this  improved  Doremus  ureometer 


FIG.    113. — Doremus- Hinds    Ure- 
ometer. • 


464  APPENDIX 

the  closed  portion  of  the  U  tube  is  filled  with  the  hypobromite  solution,  and  the  urine 
introduced  by  allowing  it  to  run  in  from  the  side  tube  by  opening  the  glass  cock 
arranged  for  that  purpose.  After  the  gas  has  risen  and  the  instrument  has  stood 
for  a  short  time  the  readings  may  be  made  in  grams  to  the  liter,  or  in  percentage. 

This  urea  determination  is  only  a  rough  clinical  one. 

Urea  Determination  by  Urease  Method. — Put  i  or  2  c.c.  of  toluol  into  each  of 
two  200  c.c.  Erlenmeyer  flasks;  to  one,  add  exactly  5  c.c.  of  a  specimen  and  TOO  c.c. 
of  distilled  water;  to  the  other,  one  Urease-Dunning  tablet,  crushed  and  dissolved 
in  about  5  c.c.  of  water,  using  a  small  glass  mortar.  Rinse  the  mortar  with  several 
portions  of  distilled  water  until  about  100  c.c.  have  been  introduced  into  second  flask 
and  then  add  exactly  5  c.c.  of  the  urine  specimen.  Stopper  both  flasks  with  cork 
and  agitate  contents.  If  time  is  not  a  consideration,  allow  flasks  to  stand  at  room 
temperature,  at  least  eight  hours,  or  use  two  tablets  and  digest  at  4o°C.  for  one  hour. 
Rapid  determination  may  be  made  by  using  but  i  c.c.  of  urine,  two  tablets  and  100 
c.c.  of  distilled  water  and  digest  between  40°  and  50° C.  for  thirty  minutes  only. 

After  the  elapse  of  proper  time,  titrate  the  two  solutions  to  a  distinct  pink  color 
with  N/io  HC1  and  methyl  orange.  The  amount  of  HC1  required  to  neutralize 
the  urease-treated  specimen,  less  the  amount  required  for  the  control,  will  give  the 
urea  content,  estimated  upon  its  equivalent  in  ammonium  carbonate. 

The  controls  of  a  large  number  of  determinations  made  at  one  time  may  be  ti- 
trated, as  to  existing  alkalinity,  with  N/io  HC1  and  methyl  orange,  one  after  another, 
in  the  same  flask,  at  the  time  the  portions  to  be  examined  for  urea  are  prepared  in 
separate  flasks.  In  such  cases,  a  sufficient  number  of  Urease-Dunning  tablets, 
for  all,  may  be  rubbed  up  in  a  mortar  with  a  measured  quantity  of  distilled  water 
and  an  aliquot  portion,  with  100  c.c.  of  distilled  water,  added  to  each  of  the  separate 
specimens  treated.  See  urease  method  for  blood. 

Gerhardt's  Test  for  Diacetic  Acid 

Add  a  few  drops  of  ferric  chloride  solution  to  10  to  50  c.c.  of  urine  as  long  as  a 
precipitate  continues  to  form.  Then  filter  and  to  the  filtrate  add  more  ferric  chloride 
solution.  A  bordeaux  red  color  shows  diacetic  acid.  The  test  is  sensitive.  As  a 
control  to  show  that  the  color  is  not  due  to  drug  elimination  (antipyrine,  salicylates, 
etc.)  boil  a  specimen  which  gave  the  test  for  three  to  five  minutes.  If  the  color  was 
due  to  drugs  it  will  be  obtained  with  a  boiled  sample  while  such  treatment  drives  off 
the  diacetic  acid.  In  Hurtley's  test  add  2.5  c.c.  HC1  and  i  c.c.  of  i%  sol.  of  sod. 
nitrate  to  10  c.c.  urine.  Shake  and  allow  to  stand  two  minutes.  Now  add  15  c.c. 
strong  ammonia  followed  by  5  c.c.  of  10%  sol.  ferrous  sulphate.  The  slow  produc- 
tion of  a  violet  color  shows  positive  test  (two  hours).  Shows  i  part  aceto-acetic 
acid  in  50,000. 

If  the  urine  shows  a  well-marked  Gerhardt  reaction  it  is  well  to  test  for  /3-oxy- 
butyric  acid. 

The  following  modification  of  Lange's  test  by  Hart  is  a  satisfactory  one,  The 
principle  involved  is  the  removal  of  acetone  and  diacetic  acid  by  heat,  then  oxidizing 
/3-oxybutyric  acid  to  acetone  with  hydrogen  peroxide  and  then  testing  for  acetone. 

Method:  Take  20  c.c.  of  urine,  dilute  with  an  equal  amount  of  water  and  add  a 
few  drops  of  acetic  acid.  Next  boil  in  a  beaker  until  the  original  amount  of  diluted 


APPENDIX  465 

urine  is  reduced  to  10  c.c.  (originally  40  c.c.).  Dilute  this  evaporated  urine  with  an 
equal  amount  of  water,  giving  us  20  c.c.  In  each  of  two  test-tubes  put  10  c.c.  of 
this  20  c.c.  To  one  tube  add  i  c.c.  of  hydrogen  peroxide  and  warm  gently,  without 
boiling,  for  one  minute;  then  cool.  The  other  tube  is  left  untreated.  Next,  to  each 
test-tube  add  10  drops  of  glacial  acetic  acid  and  5  to  10  drops  of  a  freshly  prepared 
sodium  nitroprusside  solution  and  mix.  Next  carefully  overlay  each  tube  with 
about  2  c.c.  of  concentrated  ammonia.  If  0-oxybutyric  acid  were  present  in  the 
tube  treated  with  the  hydrogen  peroxide  and  thereby  oxidized  to  acetone  a  violet- 
red  ring  will  develop  at  the  point  of  contact  while  in  the  untreated  tube  there  will 
be  no  such  color  ring. 

A  yellowish-brown  ring  from  the  presence  of  creatinin  may  show  in  the  untreated 
tube.  It  is  well  to  allow  the  tubes  to  stand  for  three  to  four  hours  before  finally 
reporting  the  absence  of  /J-oxybutyric  acid.  It  will  probably  show  0.2%. 

Acetone. — To  one-sixth  of  a  test-tube  of  urine  add  a  crystal  of  sodium  nitro- 
prusside. Make  strongly  alkaline  with  NaOH.  Shake.  The  addition  of  a  few 
drops  of  glacial  acetic  gives  a  purple  color  to  the  foam,  if  acetone  is  present. 

Acetone  is  naturally  present  in  exceedingly  small  quantity.  It  is  very  much 
increased  in  advanced  cases  of  diabetes  mellitus.  It  is  also  frequently  found  in 
scarlet  fever,  typhoid  fever,  pneumonia,  nephritis,  and  severe  anemias.  It  is  often 
present  after  ether  or  chloroform  anaesthesia. 

Although  the  following  test  can  be  applied  directly  to  the  urine  it  is  preferable 
to  obtain  distillates  when  possible  and  test  these. 

Gunning  Test. — To  about  5  c.c.  of  the  urine  add  a  few  drops  of  Lugol's  solution 
and  then  ammonium  hydrate  until  a  black  precipitate  forms.  Allow  to  stand  for 
some  time,  when,  if  acetone  is  present,  a  yellowish  precipitate  of  iodoform  crystals 
will  separate.  These  should  be  identified  by  microscope.  Iodoform  crystallizes 
in  some  modification  of  the  hexagonal  plate,  usually  a  six-pointed  stellate  form. 
If  the  crystals  are  not  clearly  defined,  throw  precipitate  on  filter,  wash  with  a  little 
water,  and  pour  over  it  a  little  hot  alcohol.  To  this  filtrate  add  water,  drop  by  drop, 
until  a  precipitate,  which  will  consist  of  well-defined  crystals  of  iodoform,  is  obtained. 
Examine  this  by  microscope. 

Never  use  an  alcoholic  solution  of  iodine  in  connection  with  this  test. 

Diazo  Reaction. — To  5  c.c.  sulphanilic  acid  solution  (sulphanilic  ac.  i  pt,  HC1 
50  pts.,  aq.  1000  pts.)  add  2  drops  of  a  0.5%  solution  of  sodium  nitrate.  Add  an 
equal  quantity  (5  c.c.)  of  urine.  Shake  and  add  quickly  2  or  3  c.c.  of  ammonium 
hydrate.  A  carmine  color,  especially  in  the  foam,  shows  a  diazo  reaction.  If  the 
reaction  is  positive,  and  the  mixture  is  allowed  to  stand  for  twenty-four  hours,  a 
precipitate  forms,  the  upper  margin  of  which  exhibits  a  green,  greenish-black  or 
violet  zone. 

Indican. — Take  10  c.c.  urine  and  treat  it  with  i  c.c.  of  sol.  of  lead  subacetate. 
Filter.  Of  this  filtrate  take  6  c.c.  and  treat  with  an  equal  amount  of  Obermayer's 
reagent;  allow  to  stand  for  five  minutes  then  shake  gently  with  2  c.c.  of  chloroform. 
Obermayer's  reagent  is  strong  HC1  containing  2  parts  of  ferric  chloride  to  the  liter 
— o.i  gram  to  50  c.c.  of  HC1. 

A  more  exact  method  is  to  pour  off  the  supernatant  acid  urine.     Wash  the  chlo- 
roform with  water,  then  pour  off  as  much  of  the  supernatant  water  as  possible 
and  add  10  c.c.  of  alcohol.     A  clear  blue  fluid  results. 
30 


466  APPENDIX 

Urobilin. — Urobilin  appears  in  considerable  quantity  in  urine  when  there  is 
much  destruction  of  red  cells,  as  in  pernicious  anaemia,  internal  haemorrhage,  and  in 
malarial  cachexia.  The  best  test  is  that  of  Schlesinger.  To  the  unfiltered  urine 
add  an  equal  amount  of  a  saturated  solution  of  zinc  acetate  in  absolute  alcohol. 
Shake,  add  a  few  drops  of  Lugol's  solution  and  filter.  Fluorescence  in  the  filtrate 
shows  the  presence  of  urobilin.  The  degree  of  blood  destruction  is  indicated  by  the 
intensity  of  the  fluorescence. 

Bile  Pigments. — A  satisfactory  test  is  that  of  Rosin  (Trousseau).  Overlay  10 
c.c.  urine  with  about  5  c.c.  of  dilute  tincture  of  iodine  (i  to  10  of  95%  alcohol). 
An  emerald  green  ring  at  the  point  of  contact  shows  the  presence  of  bile  coloring 
matter.  For  Gmelin's  lest  pass  the  urine  several  times  through  the  filter  and  then 
touch  the  paper  with  a  glass  rod  which  has  been  dipped  in  a  commercial  HNO3. 
A  green  color  play  shading  to  blue  and  violet  shows  bilirubin. 

Bile  Acids. — Bile  pigments  and  bile  acids  usually  occur  together  in  urine.  A 
very  delicate  and  reliable  test  for  bile  acids  is  that  of  Oliver.  Filter  a  specimen  of 
urine  until  it  is  quite  clear.  Then  make  reaction  acid  provided  the  urine  is  not 
acid.  The  urine  should  be  diluted  with  water  until  its  specific  gravity  is  below 
1008.  The  reagent  is  made  as  follows:  Peptone  30  grains  and  salicylic  acid  4 
grains  are  dissolved  in  8  ounces  of  distilled  water  containing  %  dram  of  acetic  acid. 

The  solution  is  filtered  until  transparent. 

For  the  test  add  20  minims  of  the  clear  acid  urine  to  60  minims  of  the  reagent. 
A  milky  turbidity  indicates  bile  acids.  If  the  turbidity  disappears  on  shaking, 
the  addition  of  more  reagent  will  cause  it  to  reappear. 

URIC  ACID  IN  URINE 

Normally  we  have  from  0.2  to  2  grams  eliminated  in  twenty-four  hours  de- 
pending on  nature  and  quantity  of  protein  intake.  Nuclein  rich  foods,  as 
sweet-breads,  liver,  etc.,  greatly  increase  the  amount. 

To  determine  the  quantity  add  to  150  c.c.  of  the  urine  50  c.c.  of  a  reagent  which 
consists  of  500  grams  ammonium  sulphate,  5  grams  of  uranium  acetate,  and  60  c.c. 
of  10%  acetic  acid  dissolved,  with  the  aid  of  heat,  in  sufficient  water  (about  700  c.c.) 
to  make  i  liter  of  solution.  The  mixture  of  urine  and  reagent  is  then  filtered.  To 
134  c.c.  of  the  filtrate  (which  represents  100  c.c.  of  the  urine),  contained  in  a  beaker, 
add  sufficient  ammonium  hydrate  to  make  strongly  alkaline,  and  let  stand  twenty- 
four  hours.  Filter,  preferably  through  hardened  filter,  wash  beaker,  and  precipitate 
thoroughly  with  10%  ammonium  sulphate  solution;  open  filter  and  with  wash  bottle 
wash  precipitate  back  into  beaker,  using  approximately  100  c.c.  of  water  to  do  so, 
add  15  c.c.  of  concentrated  sulphuric  acid,  and  then  immediately  from  burette  add 
N/20  potassium  permanganate  solution  until  a  pink  color  which  persists  for  thirty 
seconds  is  obtained.  The  number  of  cubic  centimeters  of  the  potassium  per- 
manganate solution  required  is  then  multiplied  by  0.00375;  to  this  result  add 
0.003  for  each  100  c.c.  of  filtrate  and  wash  solution  used.  The  final  result  is  the 
quantity  of  uric  acid  in  100  c.c.  of  urine. 


CHLORIDES 


These  are  normally  present  in  quantity  corresponding  to  10  to  15  grams  of  sodium 
chloride.     Diet  as  well  as  certain  pathologic  processes,  especially  the  latter,  may  cause 


APPENDIX  467 

marked  deviation  in  quantity  and  as  the  deviation  is  usually  very  well  marked, 
a  method  giving  only  fairly  accurate  results  is  sufficient  for  their  estimation. 

Dilute  5  c.c.  of  the  urine,  which  should  be  free  from  albumin,  with  50  to  75  c.c. 
of  distilled  water,  add  10  to  15  drops  of  a  solution  of  potassium  chromate  and  then 
from  a  burette  add  N/io  AgNO3  until  a  very  slight  pinkish  tinge  is  obtained. 
The  number  of  cubic  centimeters  of  AgN03  required,  multiplied  by  0.00585  will 
give  the  quantity  of  sodium  chloride  to  which  the  chlorine  in  5  c.c.  of  urine  is 
equivalent.  Where  a  considerable  degree  of  accuracy  is  demanded  one  should  use 
either  the  Arnold  or  the  Harvey  modification  of  Volhardt's  method. 

FORMALDEHYDE  IN  URINE  AFTER  ADMINISTRATION  OF  UROTROPIN 

As  formaldehyde  fails  to  appear  in  the  urine  of  possibly  52%  of  those  taking 
urotropin  as  a  geni to-urinary  antiseptic  in  quantities  sufficient  to  inhibit  bacterial 
growth  (1-5000)  the  usual  tests  are  too  delicate.  As  a  practical  guide  to  efficient 
breaking  up  of  urotropin  the  test  proposed  by  Burnam  is  to  be  recommended. 

To  about  10  c.c.  urine  in  a  test-tube  at  body  temperature  add  3  drops  of  Y^% 
solution  of  phenylhydrazin  hydrochloride  and  3  drops  of  a  5%  solution  of  sodium 
nitroprusside.  Finally  allow  a  few  drops  of  20%  solution  of  NaOH  to  run  down 
the  side  of  the  test-tube  and  as  this  diffuses  throughout  the  urine  a  deep  purplish-blue 
color,  rapidly  changing  to  a  dark  green  becoming  lighter  green  and  finally  pale 
yellow  will  show  if  formaldehyde  is  being  excreted  in  sufficient  strength.  In  the 
absence  of  sufficient  formaldehyde  a  reddish  color  develops  which  finally  turns  to  a 
light  yellow. 

In  order  to  obtain  effect  from  urotropin  it  is  necessary  to  have  the  urine  acid. 
This  is  best  accomplished,  if  an  acid  reaction  is  absent,  by  the  administration  of 
dihydrogen  sodium  phosphate  (acid  sodium  phosphate).  In  carrying  out  Burnam 
test  albumin  in  the  urine  confuses  the  color  reaction.  By  careful  boiling  to 
precipitate  the  albumin,  then  filtering,  we  avoid  confusing  colors. 

Phenolsulphonephthalein  Test  for  Renal  Efficiency 

.  Geraghty  has  recently  stated  that  in  35  cases  where  an  autopsy  made  it  possible 
to  verify  the  accuracy  of  this  test  the  lesions  revealed  at  autopsy  corresponded 
closely  with  the  results  of  the  test.  Again  in  30  nephrectomies  the  conditions 
found  were  in  accordance  with  the  results  of  the  test.  The  general  opinion  of 
those  who  have  used  the  test  is  that  it  is  more  reliable  than  cryoscopy  and  far  easier 
of  application.  The  technic  is  as  follows:  One  c.c.  of  the  phthalein  solution 
containing  6  mg.  is  injected  intramuscularly  or  subcutaneously.  The  drug  can  be 
bought  in  ampules  ready  for  use.  About  twenty  minutes  before  injecting  the  drug 
the  patient  is  given  from  200  to  400  c.c.  of  water  to  drink.  After  the  injection  the 
bladder  is  emptied  with  a  catheter  and  the  time  is  accurately  noted  when  the  urine 
which  subsequent  to  the  emptying  of  the  bladder  and  being  allowed  to  drop  into  a 
test-tube  containing  i  drop  of  a  25%  sodium  hydrate  solution  first  shows  a  pinkish 
tinge.  This  is  recorded  as  the  time  of  appearance  of  the  drug  in  the  urine  and 
normally  is  about  ten  minutes.  The  catheter  is  then  withdrawn  and  the  urine  that 
is  passed  in  the  first  hour  collected  and  subsequently  that  passed  in  the  second  hour. 


468  APPENDIX 


irple- 


To  each  hour's  specimen  sufficient  25%  sodium  hydrate  is  added  to  give  a  pui 
red  colcr  and  the  entire  amount  is  then  poured  into  a  liter  flask  and  made  up  to 
looo  c.c.     A  similar  treatment  is  employed  for  the  urine  of  the  second  hour.     The 
amount  of  drug  eliminated  in  each  hour  is  then  determined  by  a  colorimeter. 

Cabot  has  proposed  the  use  of  a  series  of  10  test-tubes  containing  solutions  of 
the  drug  representing  from  5%  to  50%  of  the  drug  dose,  each  tube  containing  5% 
more  than  the  preceding  one.  These  comparison  solutions  may  be  made  up  with 
the  patient's  urine  obtained  at  the  time  of  emptying  the  bladder  so  that  the  con- 
fusion which  may  obtain  when  water  is  used  is  avoided.  It  has  recently  been  pro- 


FIG.  114. — Rowntree  and  Geraghty  modification  of  Hellige  Colorimeter. 

posed  to  make  the  standards  with  water  and  use  a  piece  of  yellow  glass  for  match- 
ing. The  urine  to  be  tested  made  up  to  1000  c.c.  as  previously  described  is  then 
poured  into  a  test-tube  of  similar  size  and  matched. 

In  normal  cases  Cabot  got  46%  of  the  drug  eliminated  in  the  first  hour,  the 
average  for  the  second  hour  being  17%.  The  quantity  of  urine  secreted  in  eithei 
hour  has  no  relation  to  the  test,  which  is  the  percentage  of  drug  eliminated.  In 
cases  with  serious  kidney  disease  the  amount  of  drug  eliminated  in  the  first  hour 
may  range  from  5  to  12%. 

When  the  question  of  the  kidney  involved  arises,  the  urine  must  be  taken  by 
ureteral  catheterization  or  by  a  separator. 

Dunning  has  devised  a  simple  inexpensive  colorimeter  for  the  drug  excretion 


APPENDIX 


469 


determination  in  place  of  the  more  expensive  colorimeters  such  as  those  of  Duboscq 
and  Hellige.  A  cut  of  the  apparatus  is  given.  As  a  rule,  I  make  up  my  known 
standards  with  urine  instead  of  water  as  giving  better  comparison  and  pour  a  darker 
known  solution  from  a  graduated  cylinder  into  a  Nessler  jar  as  described  under 
blood-sugar  estimations. 


FIG.  115. — Dunning  colorimeter. 


F— CHEMICAL  EXAMINATION  OF  GASTRIC  CONTENTS 

The  test  breakfast  ordinarily  used  is  that  of  Ewald  (one  shredded  wheat  biscuit 
or  two  small  pieces  of  toast  with  400  c.c.  of  water  is  what  is  usually  given).  This 
Ewald  breakfast  is  a  low-grade  stimulant  to  acid  production.  It  is  given  in  the 
morning  on  an  empty  stomach.  If  at  supper,  the  night  before,  the  patient  partake 
of  raspberry  jam  the  finding  of  the  characteristic  seeds  in  the  stomach  contents 
the  next  morning  would  be  evidence  of  lack  of  motor  activity.  The  Fischer  meal 
which  contains  a  4-ounce  Hamburg  steak  in  addition  to  the  water  and  toast  of  the 
Ewald  is  withdrawn  after  three  hours.  v 

The  stomach  tube  is  more  easily  passed  if  it  be  thoroughly  chilled  in  ice  water 
without  the  use  of  any  lubricant. 

The  stomach  tube  should  be  passed  one  hour  after  the  Ewald  breakfast  and  if 
more  than  50  c.c.  of  fluid  be  obtained  it  indicates  stasis  or  hypersecretion. 

Free  HCL— Filter  the  gastric  contents  and  test  first  for  free  HC1.  The  most 
reliable  and  sensitive  test  is  that  of  Gunsberg.  The  reagent,  which  should  be 
freshly  prepared,  consists  of  phloroglucin  3  grams,  vanillin  i  gram,  and  absolute 
alcohol  30  c.c.  By  mixing  2  drops  of  gastric  juice  and  an  equal  quantity  of  Gunsberg 
reagent  in  a  small  porcelain  dish  and  carefully  heating  above  a  flame  we  obtain  a 
carmine  red  color  if  free  HC1  be  present.  A  water-bath  is  preferable. 

The  Gluzinski  test  is  of  value  in  the  diagnosis  of  gastric  cancer.    In  this,  three 


470  APPENDIX 

tests  for  free  HC1  are  made,  one  from  gastric  contents  and  washings  taken  in  the 
morning  before  taking  any  form  of  food.  Then  give  the  albumin  of  2  hard  boiled 
eggs,  finely  minced  in  150  c.c.  water.  One  hour  later  remove  the  contents  and  ex- 
amine for  free  HC1.  Then  give  a  meal  of  broth,  lightly  broiled  steak,  mashed 
potatoes  and  bread  and  three  hours  later  remove  contents  with  stomach  tube  and 
test  again  for  free  HC1. 

For  lactic  acid  a  modification  of  Strauss'  method  is  quite  satisfactory.  Shake, 
in  a  test-tube,  5  c.c.  of  gastric  contents  with  20  c.c.  of  ether,  allow  to  settle  and 
pour  off  5  c.c.  of  the  supernatant  ether  into  another  test-tube.  To  this  ether  add 
20  c.c.  of  water  and  2  drops  of  a  i  to  9  solution  of  ferric  chloride  and  shake  well. 
The  presence  of  i%  of  lactic  acid  will  give  an  intense  greenish  color. 

Total  Acidity. — Having  determined  the  presence  or  absence  of  free  hydrochloric 
or  lactic  acid,  we  should  make  a  quantitative  test  of  the  various  determinations  of 
the  acidity  of  gastric  juice  (a  modified  Topfer  test).  These  are:  i.  Free  HC1.  2. 
Combined  HC1.  3.  Acid  salts,  and  4.  Total  acidity. 

To  10  c.c.  of  filtered  gastric  contents,  in  a  beaker,  add  3  drops  of  dimethyl-amido- 
azo-benzol  solution  (a  1/2%  solution  in  95%  alcohol).  In  the  presence  of  "free 
HC1  the  fluid  becomes  a  rich  carmine  pink. 

After  reading  the  burette  run  in  N/io  NaOH  solution  until  the  pink  color  is 
discharged  and  a  light  yellow  color  is  obtained.  This  reading  multiplied  by  10 
gives  the' amount  of  free  HC1  in  degrees,  a  degree  corresponding  to  i  c.c.  N/io 
NaOH.  Next  add  6  drops  of  a  1/2%  alcoholic  solution  of  phenolphthalein  to  the 
light  yellow  fluid  in  the  beaker.  Again  titrating  the  same  preparation  we  add 
N/io  NaOH  until  a  faint  but  distinct  pink  color  is  produced.  The  number  of 
cubic  centimeters  added  for  the  free  HC1  plus  the  number  to  give  the  pink  color 
when  multiplied  by  10  gives  the  total  acidity  in  degrees.  (For  example:  2.5  c.c. 
N/io  NaOH  used  to  obtain  yellow  color — 2.5X10  =  25  or  acidity  due  tofreeHCl. 
After  adding  the  phenolphthalein,  4  c.c.  N/io  NaOH  required  to  produce  pink 
color — 4+2.5X10  =  65  or  total  acidity  in  terms  of  acidity.  This  means  that  it 
would  require  65  c.c.  N/io  NaOH  to  neutralize  100  c.c.  of  gastric  juice.  A  total 
acidity  of  60  is  about  normal.  To  obtain  percentage  in  HC1  multiply  by  0.00365; 
thus,  65X0.00365  =  0.23%  HC1.) 

Having  determined  the  total  acidity  add  3  c.c.  of  10%  neutral  calcium  chloride 
solution  to  the  gastric  contents  already  in  the  beaker.  As  a  result  of  the  formation 
of  acid  calcium  phosphate  the  pink  color  is  discharged.  Again  add  N/io  NaOH 
from  the  burette  until  the  pink  color  is  restored.  The  number  of  cubic  centimeters 
used  gives  the  amount  of  acid  salts  present. 

From  the  figures  for  the  total  acidity  subtract  the  sum  of  that  for  free  HC1  and 
for  acid  salts  and  the  remainder  will  give  the  acidity  due  to  combined  HC1. 


WOLFF  AND  JUNGHANS'  TEST 

This  is  an  important  test  as  distinguishing  benign  achylias  from  those  associated 
with  malignant  disease  of  the  stomach.  Of  course,  where  the  carcinoma  originates 
in  the  lesion  of  an  ulcer  or  when  small  or  in  the  region  of  the  pylorus  there  may  not 
be  an  achylia,  free  HC1  being  present. 

While  interpretation  of  increased  albumin  in  those  cases  of  carcinoma  showing 


APPENDIX  471 

free  HC1,  owing  to  digestive  action,  would  be  difficult,  this  is  less  so  in  the  typical 
case  with  its  absent  free  HC1.  To  carry  out  the  test  it  is  best  to  give  the  patient 
only  a  light  supper  and  follow  this  with  mild  cathartic  the  same  evening.  Give 
the  Ewald  test  breakfast  the  next  morning  and  withdraw  contents  in  one  hour.  Fil- 
ter the  contents  and  of  the  nitrate  deposit  i  c.c.,  0.5  c.c.,  0.25  c.c.,  o.i  c.c.,  0.05  c.c., 
and  0.025  c.c.  in  six  test-tubes.  Make  up  contents  of  each  tube  to  10  c.c.  with 
distilled  water.  The  dilutions  will  be  from  i  to  10  in  the  first  tube  to  i  to  400  in  the 
sixth  tube.  After  mixing  the  tubes  superimpose  i  c.c.  of  the  reagent  on  each  tube. 
The  reagent  is  phosphotungstic  acid  0.3  gram,  concentrated  HC1  i  c.c.,  96%  alcohol 
20  c.c.,  distilled  water  to  make  200  c.c. 

Normal  gastric  juice  gives  a  turbid  albumin  ring  in  the  first  two  or  occasionally 
in  the  third  (i  to  40)  tube.  Turbidity  in  the  i  to  100  or  over  is  suggestive  of  car- 
cinoma. 

The  increase  of  albumin  content  in  malignant  achylias  is  thought  to  be  due  to 
the  action  of  a  specific  ferment  capable  of  acting  on  the  protein  content  of  the 
meal  forming  the  soluble  albumin  we  test  for. 


PEPSIN 

The  determination  of  the  quantity  of  pepsin  is  rather  troublesome  by  the  older 
methods,  and  in  consequence  a  simple  method,  which  also  uses  a  protein,  will  be  given. 
The  protein  used  is  edestin,  a  globulin  obtained  from  hempseed.  Dissolve  o.i 
gram  of  the  edestin  in  100  c.c.  of  0.1%  HC1.  (This  is  most  quickly  done  by 
sprinkling  the  edestin  over  the  surface  of  the  acid.)  In  each  of  six  small  test-tubes 
place  exactly  2  c.c.  of  the  edestin  solution  (thus  giving  2  mg.  of  the  substance 
to  each  tube).  Dilute  i  c.c.  of  stomach  contents  to  20  c.c.  with  water.  To  the  above- 
mentioned  test-tubes  are  added,  respectively,  0.2,  0.4,  0.6,  0.8,  and  i  c.c.  of  the 
diluted  sample,  the  last  test-tube  being  left  as  a  blank.  Shake  each  test-tube  so 
that  its  contents  will  be  mixed.  Allow  the  tubes  to  stand  for  thirty  minutes.  Then 
to  each  tube  add  0.3  c.c.  of  a  saturated  solution  of  NaCl.  In  those  tubes  which  do 
not  contain  sufficient  pepsin  to  digest  2  mg.  of  edestin  a  white  cloud  or  precipitate 
will  be  produced  while  all  others  remain  clear.  (The  blank  or  sixth  tube  must  give 
a  copious  precipitate  or  the  edestin  solution  is  worthless.)  The  tube  containing  the 
smallest  quantity  of  the  dilute  sample  and  giving  no  cloud  or  precipitate  is  selected. 
Then  by  the  formula  2  x  dilution  divided  by  the  volume  of  dilute  sample  present  is 
obtained  the  number  of  milligrams  of  edestin  that  will  be  digested  by  i  c.c.  of  the 
undiluted  sample.  This  quantity  normally  is  said  to  be  100.  If  all  but  the  sixth 
tube  failed  to  give  the  cloud  or  precipitate  a  greater  dilution  of  the  sample  must  be 
made  and  the  test  repeated.  The  edestin  solution  rapidly  decomposes  and  must 
be  made  fresh  when  needed. 

Mett's  Method  for  Quantitative  Estimation  of  Pepsin. — Capillary  glass  tubes, 
i  or  2  mm.  in  diameter  are  filled  with  white  of  egg,  and  after  plugging  one  end  with 
bread  crumbs  the  tubes  are  placed  in  water  at  97°  or  98°C.  for  five  minutes.  The 
tubes  are  sealed  with  plasticine  or  sealing  wax.  For  the  test  dilute  i  c.c.  of  gastric 
juice  with  15  c.c.  N/20  HC1  and  put  some  into  a  test-tube.  Then  file  off  two  2  cm. 
columns  of  the  capillary  tube  with  coagulated  egg  albumin.  Place  test-tube  with  its 
albumin  tubes  in  incubator  for  twenty-four  hours.  Then  measure  the  digested  ends 


472 


APPENDIX 


of  the  two  capillary  tubes  and  take  average.  The  square  of  this  figure  gives 
number  of  units  of  pepsin  in  the  i  to  16  dilution  of  gastric  juice  and  when  multi- 
plied by  1 6  the  number  of  units  in  i  c.c.  of  gastric  juice.  Normal  variation  is 
8  to  100  units. 

G— CHEMICAL  EXAMINATION  OF  DUODENAL  JUICE 

The  general  discussion  of  the  subject  of  the  examination  of  the  duodenal  juice  is 
taken  up  in  Part  IV.  As  stated  there  the  lipase  tests  are  rather  unreliable  so  only 
tests  for  amylase  (amylopsin)  and  protease  (trypsin),  as  recommended  by  Myers 
and  Fine  are  given. 

Wohlgemuth  Method  for  Amylase. — Place  5  c.c.  of  i%  soluble  starch  solution  in 
each  of  six  small  test-tubes.  Tube  I  serves  as  a  control,  and  to  each  of  the  other  five 
tubes  add  0.05,  o.i,  0.25,  0.5  and  i.o  c.c.  of  the  duodenal  juice  diluted  one  half  with 
distilled  water. 

Place  in  37°C.  incubator  for  one-half  hour,  add  cold  water  to  almost  fill  the  tubes 
and  then  several  drops  of  N/io  iodine.  The  positive  tube  will  show  an  entire  ab- 
sence of  blue  color.  The  amylase  strength  is  represented  by  the  number  of  cubic 
centimeters  of  starch  solution  i  c.c.  of  undiluted  juice  can  digest.  If  i  c.c.  of  un- 
diluted duodenal  juice  digests  5  c.c.  of  starch  solution  the  amylase  strength  is  5;  if 
o.i,  50.  The  average  normal  is  about  50. 

Gross  Casein  Method  for  Trypsin. — Put  in  each  of  six  test-tubes  5  c.c.  of  o.i  %  solu- 
tion of  casein  in  o.i  %  sodium  carbonate  solution  and  add  to  each  one,  except  tube  I, 
which  serves  as  control,  the  same  amount  of  diluted  duodenal  juice  as  given  above 
for  amylase.  Incubate  for  fifteen  minutes  at  38°C.  and  acidify  with  a  few  drops  of 
dilute  acetic  acid.  The  activity  is  calculated  as  for  amylase  and  is  normally  from 
4  to  10.  White  makes  a  i%  dilution  of  the  duodenal  fluid  with  water  and  puts  10 
c.c.  of  0.1%  casein  solution  in  each  tube.  The  tube  with  o.i  c.c.  of  diluted  fluid 
would  therefore  be  a  i  to  10,000  dilution  and  that  with  i  c.c.  a  i  to  1,000.  Nor- 
mally the  i  to  10,000  tube  remains  cloudy.  Clouding  of  the  i  to  1,000  tube  would 
show  marked  lowering  of  tryspin. 

H— DISINFECTANTS  AND  INSECTICIDES 

By  disinfection  is  meant  the  destruction  of  injurious  bacteria. 

Sterilization  is  where  all  living  things  are  destroyed. 

Germicides  are  substances  which  kill  bacteria  while  antiseptics  are  those  which 
are  inimical  to  the  growth  of  bacteria. 

Formalin  is  antiseptic  in  1-50,000  dilution  but  germicidal  only  in  1-20. 

Deodorants  may  or  may  not  be  antiseptic  or  germicidal.  An  insecticide  may 
or  may  not  be  a  germicide  and  vice  versa. 

In  disinfection  we  must  consider 

(1)  Strength  of  solution.     It  must  always  be  kept  in  mind  that  the  strength  of 
a  germicide  solution  when  added  to  an  equal  amount  of  material  to  be  disinfected 
is  reduced  in  strength  one-half.     Thus  i  pint  of  a  5%'comp.  cresol  solution  added  to 
i  pint  of  faecal  material  has  only  a  2^%  disinfecting  effect. 

(2)  Time  of  application.     A  common  mistake  is  to  consider  a  few  minutes  as  suffi- 
cient for  contact  of  germ-containing  material  with  the  disinfectant.    In  the  faeces- 


APPENDIX  473 

cresol  mixture  above  noted  that  action  of  the  disinfectant  should  continue  at  least 
one  hour  before  emptying  the  vessel. 

(3)  Nature  of  medium  in  which  disinfectant  acts.     Bacteria  are  much  more  re- 
sistant to  germicide  agents  when  contained  in  solution  rich  in  organic  matter  than 
when  suspended  in  pure  water.     Thus  Liq.  Cresol.  Comp.  has  a  phenol  coefficient 
of  3  without  organic  matter  and  only  1.87  with  organic  matter. 

(4)  Temperatures.     Disinfecting  solutions  show  greater  power  as  the  temperature 
rises,  and  act  less  efficiently  in  the  cold.     A  body  temperature  (3Q°C.)  is  a  good 
one. 

By  Coefficient  of  Inhibition  we  mean  time  and  concentration  necessary  to  prevent 
development  of  bacteria. 

By  Inferior  Lethal  Coefficient  we  mean  time  and  concentration  necessary  to  kill 
nonspore-bearing  bacteria. 

By  Superior  Lethal  Coefficient  we  mean  time  and  concentration  necessary  to 
kill  spore-bearing  bacteria. 

U.  S.  Hygienic  Laboratory  Phenol  Coefficient. — In  determining  the  strength  of  a 
disinfectant  it  is  compared  with  that  of  phenol  (commercial  carbolic  acids  vary  in 
their  phenol  content).  If  more  powerful  than  phenol  the  coefficient  will  be  greater 
than  i.  In  the  U.  S.,  disinfectants  are  rated  according  to  the  "Hygienic  Laboratory 
Phenol  Coefficient." 

The  determinations  when  organic  matter  was  not  present,  were  conducted  as 
follows : 

"The  experiments  were  done  at  a  temperature  of  2o°C.  maintained  by  means  of  a 
water-bath.  The  quantity  of  each  dilution  of  disinfectant  and  phenol  used  in  each 
experiment  was  5  c.c.  The  culture  used  was  B.  typhosus,  twenty-four-hour 
extract  broth,  filtered.  The  seeding  tubes  containing  the  disinfectant  dilutions  and 
the  filtered  broth  culture  of  B.  typhosus  were  placed  in  the  water-bath  and  allowed 
to  reach  the  temperature  of  2o°C.  before  starting  the  experiment. 

"  The  seeding  tubes  were  inoculated  successively  every  fifteen  seconds  with  o.i  c.c. 
of  the  typhoid  culture.  Each  tube  was  gently  shaken  after  it  was  inoculated.  At 
the  end  of  each  two  and  one-half  minute  period,  for  fifteen  minutes,  plants  were 
made  from  the  seeding  tubes  into  tubes  of  extract  broth.  The  medium  used  was 
standard  extract  broth  having  a  reaction  of  +1.5.  The  quantity  of  broth  in  each 
tube  was  approximately  10  c.c. 

"The  tubes  were  incubated  at  37°C.  for  forty-eight  hours,  at  the  end  of  which 
the  results  were  recorded. 

"  To  determine  the  coefficient,  the  figure  representing  the  degree  of  dilution  of  the 
weakest  strength  of  the  disinfectant  that  kills  within  two  and  one-half  minutes  is 
divided  by  the  figure  representing  the  degree  of  dilution  of  the  weakest  strength  of 
the  phenol  control  that  kills  within  the  same  time.  The  same  is  done  for  the 
weakest  strength  that  kills  within  .fifteen  minutes.  The  mean  of  the  two  is  the 
coefficient. 

"  When  the  determinations  were  made  as  to  efficiency  in  the  presence  of  organic 
matter  a  stock  solution  of  10%  peptone  and  5%  gelatin  in  water  was  made  up  and 
of  this  i  c.c.  was  put  in  a  tube  and  then  inoculated  with  o.i  c.c.  of  typhoid  culture. 
Then  4  c.c.  of  the  varying  phenol  and  disinfectant  dilutions  were  added  and  the 
tests  conducted  as  above." 


474  APPENDIX 

Disinfectants  may  be  (A)  Physical,  (B)  Gaseous,  (C)  Chemical 

(A)  Of  the  physical  disinfectants  we  have 

(1)  Sunlight.     The  red  and  yellow  rays  practically  inert.     The  violet  and  ultra 
violet  most  active.     Direct  sunlight  kills  plague  bacilli  in  less  than  one  hour — 
typhoid  bacilli  in  six. 

(2)  Burning.     Very  efficient  but  expensive. 

(3)  Boiling.     Especially  in  carbonate  of  soda  solution  for  about  one  hour  is  a 
very  efficient  disinfectant.     Nonspore-bearing  bacteria  are  killed  almost  instantly 
by  a  boiling  temperature.     One  must  remember  that  the  boiling  temperature  is 
lower  at  mountainous  elevations. 

(4)  Steam.     Extremely  efficient.     The  condensation  of  the  steam  on  the  object 
to  be  sterilized  gives  off  latent  heat  and  produces  a  vacuum. 

(B)  Of  the  gaseous  disinfectants  we  have  the  very  efficient  germicide  formal- 
dehyde gas  and  the  weakly  gerrnicidal,  but  potent  insecticide,  sulphur  dioxide. 

Formaldehyde  gas  is  practically  valueless  as  an  insecticide. 

Bromine,  chlorine  and  hydrocyanic  acid  gas  have  a  certain  degree  of  efficiency 
but  are  not  of  practical  application.  Hydrocyanic  acid  gas  is  especially  dangerous 
on  account  of  its  extreme  toxicity. 

(i)  Formalin. — This  is  a  40%  solution  of  formaldehyde  gas,  but  is  as  a  rule 
of  less  strength  from  evaporation  or  otherwise.  Formaldehyde  is  efficient  as  a 
surface  disinfectant  when  the  temperature  is  above  5o°F.  and  the  air  contains  at 
least  60%  of  moisture.  It  is  not  efficient  in  cold  dry  rooms.  Owing  to  its  lack 
of  penetrating  power  it  is  not  efficient  for  the  disinfection  of  mattresses,  or  similar 
articles.  To  prepare  a  room  for  disinfection  we  must  measure  the  cubic  space  to 
ascertain  the  necessary  amount  of  formalin  to  use  and  stuff  up  or  better  paste 
up  with  newspaper  all  cracks  and  openings. 

In  the  production  of  formaldehyde  gas  the  more  expensive  autoclaves  and  lamps 
have  largely  been  replaced  by  the  simple  formalin  permanganate  method.  In  this 
one  pours  500  c.c.  of  formalin  on  250  grams  of  potassium  permanganate  for  each 
1000  cubic  feet  with  six  to  twelve  hours'  exposure. 

In  employing  this  method,  take  a  pan  partly  filled  with  water.  Place  in  this 
a  second  metal  or  glass  receptacle  containing  the  permanganate.  Then  pour  the 
formalin  on  the  permanganate  crystals.  The  gas  is  generated  in  great  amount  in 
a  few  seconds.  The  receptacle  containing  the  permanganate  and  formalin  should 
be  large  enough  to  contain  10  times  the  volume  of  formalin,  as  there  is  a  tendency 
for  the  mixture  to  foam  over  the  sides  of  the  dish. 

Another  practical  method  is  the  fonnalin-sheet-spraying  one.  The  formalin 
(40%)  should  be  sprayed  on  sheets  suspended  in  the  room  in  such  a  manner  that 
the  solution  remains  in  small  drops  on  the  sheet.  Spray  not  less  than  10  ounces 
of  formalin  (40%  formaldehyde)  for  each  1000  cubic  feet.  Used  in  this  way  a 
sheet  will  hold  about  5  ounces  without  dripping  or  the  drops  running  together. 
The  room  must  be  very  tightly  sealed  in  disinfecting  with  this  process  and  kept 
closed  not  less  than  twelve  hours.  The  method  is  limited  to  rooms  or  apartments 
not  exceeding  2000  cubic  feet.  The  formalin  may  also  be  sprayed  upon  the  walls, 
floors,  and  objects  in  the  room. 

Paraform  Lamps. — For  single  rooms  the  use  of  the  paraform  lamp  is  quite  con- 
venient. Special  lamps  can  be  obtained  to  burn  the  paraform  tablets  or  a  pint 


APPENDIX  475 

tincup  will  suffice  for  the  heating  of  i  ounce  of  paraform.  The  lamp  or  alcohol 
flame  under  the  receptacle  must  not  be  high  enough  to  ignite  the  paraform  which 
burns  readily  and  in  so  doing  does  not  give  off  formaldehyde  gas.  One  ounce  of 
paraform  is  sufficient  for  a  space  of  500  cubic  feet.  One  can  dissolve  2  ounces  of 
paraform  in  8  ounces  of  boiling  water  and  then  pour  .this  over  4  ounces  of  potassium 
permanganate  in  a  2-gallon  pail. 

N.  Y.  Health  Department  Method.— After  a  prolonged  series  of  tests  the  N.  Y. 
Department  of  Health  gave  preference  to  the  following  formula: 

Paraformaldehyde  30  grams,  potassium  permanganate  75  grams,  water  90 
grams.  The  chemicals  are  mixed  in  a  deep  quart  pan  and  the  water  is  added  and 
the  mixture  stirred.  The  evolution  of  gas  is  slow  in  starting  but  is  complete  in 
five  to  ten  minutes. 

It  was  found  that  87%  of  the  gas  was  evolved  and  the  quantities  given  above 
suffice  to  disinfect  1000  cubic  feet  in  four  hours.  It  is  well  to  put  the  small  pan 
containing  the  chemicals  in  a  larger  one  to  prevent  danger  of  fire  and  soiling  of  the 
floor  by  the  frothing  of  the  mixture. 

Sulphur  Dioxide. — Sulphur  dioxide  is  fairly  efficient,  but  requires  the  presence 
of  moisture.  It  is  only  a  surface  disinfectant  and  is  lacking  in  penetrating  proper- 
ties. An  atmosphere  containing  4.5%  can  be  obtained  by  burning  5  pounds  of 
sulphur  per  1000  cubic  feet  of  space.  This  amount  requires  the  evaporation  or 
volatilization  of  about  i  pint  of  water.  Under  these  conditions  the  time  of  ex- 
posure should  be  not  less  than  twenty-four  hours  for  bacterial  infections.  A  shorter 
time  will  suffice  for  fumigation  necessary  to  kill  mosquitoes  and  other  vermin.  Dry 
sulphur  dioxide  produced  by  burning  2  pounds  of  sulphur  for  each  1000  cubic  feet 
of  space  will" answer  for  this  purpose.  An  exposure  of  from  two  to  three  hours  is 
sufficient. 

The  sulphur  may  be  burned  in  shallow  iron  pots  (Dutch  ovens),  containing  not 
more  than  30  pounds  of  sulphur  for  each  pot,  and  the  pots  should  stand  in  vessels 
of  water.  The  sulphur  pots  should  be  elevated  from  the  bottom  of  the  compart- 
ment to  be  disinfected  in  order  to  obtain  the  maximum  possible  percentage  of  com- 
bustion of  sulphur.  The  sulphur  should  be  in  a  state  of  fine  division,  and  ignition 
is  best  accomplished  with  alcohol  (special  care  being  taken  with  this  method  to 
prevent  damage  to  cargo  or  vessel  by  fire),  or  the  sulphur  may  be  burned  in  a  special 
furnace,  the  sulphur  dioxide  being  distributed  by  a  power  fan.  This  method  is 
peculiarly  applicable  to  cargo  vessels. 

Liquefied  sulphur  dioxide  may  be  used  for  disinfection  in  place  of  sulphur  dioxide 
generated  as  above,  it  being  borne  in  mind  that  this  process  will  require  2  pounds 
of  the  liquefied  gas  for  each  pound  of  sulphur,  as  indicated  in  the  above  paragraphs. 

Sulphur  dioxide  is  especially  applicable  to  the  holds  of  vessels  or  to  apartments 
that  may  be  tightly  closed  and  that  do  not  contain  objects  that  would  be  injured 
by  the  gas.  Sulphur  dioxide  bleaches  fabrics  or  materials  dyed  with  vegetable  or 
aniline  dyes.  It  destroys  linen  or  cotton  goods  by  rotting  the  fiber  through  the 
agency  of  the  acids  formed.  It  injures  most  metals.  It  is  promptly  destructive  of 
all  forms  of  animal  life.  This  property  renders  it  a  valuable  agent  for  the  extermi- 
nation of  rats,  insects,  and  other  vermin.  Sulphur  dioxide  is  a  germicide  only  in  the 
presence  of  moisture,  and  even  then  will  not  kill  spore-bearing  organisms.  If  'cloth- 
ing is  washed  immediately  after  sulphur  disinfection  the  rotting  effect  will  be  greatly 


476  APPENDIX 

lessened.     If  used  in  spaces  containing  machinery  all  metal  parts  should  be  coa 
with  vaseline. 

CHEMICAL  SOLUTIONS 


Bichloride  of  mercury  is  usually  sold  in  the  form  of  antiseptic  tablets.  As  a 
disinfectant. for  the  infectious  diseases  it  is  usually  used  in  a  strength  of  i-iooo. 
The  solution  should  be  made  in  a  wooden  or  earthenware  vessel.  As  bichloride  forms 
inert  albuminates  it  should  not  be  used  in  the  disinfection  of  sputum,  faeces  or  any 
albuminous  excreta.  It  must  be  remembered  that  bichloride  is  a  mordant  so  that 
any  stains  in  soiled  clothing  will  remain  permanent.  For  disinfection  of  clothing 
the  material  should  be  left  in  i-iooo  bichloride  for  one  hour.  Dishes  for  food 
should  never  be  disinfected  in  bichloride  on  account  of  the  danger  from  poisoning. 
Floors  and  walls  may  be  disinfected  with  i-iooo  bichloride  applied  with  a  mop. 
Allow  the  solution  to  dry  on  the  floor  or  walls. 

Formalin. — A  5%  solution  of  commercial  formalin  in  water  (50  c.c.  formalin 
950  c.c.  water)  makes  a  satisfactory  disinfectant  for  soiled  clothing.  It  is  also 
valuable  for  albuminous  material.  The  disinfectant  must  act  in  a  strength  of  5% 
so  that  if  i  pint  of  fasces  is  to  be  disinfected  we  should  add  i  pint  of  a  10%  formalin 
solution  and  allow  it  to  act  for  one  hour. 

Carbolic  Acid. — It  is  soluble  in  water  to  the  extent  of  about  5%  and  in  such 
strength  it  is  an  efficient  disinfectant.  The  solution  should  be  made  with  hot  water. 

In  standardizing  disinfectants  carbolic  acid  is  used  as  the  standard.  It  how- 
ever is  expensive  and  there  is  often  difficulty  in  making  up  satisfactory  solutions. 
More  efficient  and  more  convenient  is  the  Liquor  cresolis  comp.  U.  S.  P.  This  may 
be  prepared  by  mxing  up  equal  parts  of  cresol  and  soft  soap  as  noted  on  page  13. 
This  has  a  value  according  to  tests  made  in  the  Hygienic  Laboratory  of  3,  making 
it  in  tests  without  organic  matter  three  times  as  efficient  as  carbolic  acid.  Under 
similar  conditions  lysol  had  a  value  of  2.12,  creolin  3.25  and  trikresol  of  2.62. 

Equal  parts  of  a  5%  solution  of  Liq.  Cresol.  Comp.  and  the  fasces,  urine  or 
sputum  to  be  disinfected  is  satisfactory  for  disinfection  provided  the  mixture  is 
allowed  to  stand  for  one  hour.  Here  we  would  have  the  effect  of  a  2^%  solution. 
Liq.  Cresol.  Comp.  (5%)  is  an  excellent  disinfectant  for  contaminated  bedclothing, 
etc.  It  is  also  most  suitable  for  the  disinfection  of  floors  and  walls. 

Sulphate  of  Copper. — This  salt  has  a  remarkable  effect  on  certain  species  of  algae 
so  that  in  strengths  of  i  to  1,000,000  it  is  destructive.  In  i  to  400,000  it  will  kill 
typhoid  bacilli  in  twenty-four  hours  in  water  that  is  not  too  full  of  organic  matter. 

Hydrogen  Dioxide. — A  2%  solution  will  kill  anthrax  spores  in  three  hours.  It 
is  useful  in  treatment  of  anaerobic  infections,  as  gas  bacillus  ones. 

Chinosol. — This  is  a  derivative  of  quinoline,  a  coal-tar  product.  It  is  a  yellow 
powder  readily  soluble.  It  does  not  coagulate  albumin  and  leaves  no  stain  on 
clothing.  It  is  efficient  in  a  strength  of  i  to  500  or  i  to  1000. 

Lime. — It  must  be  remembered  that  air-slaked  lime  is  inert  as  a  disinfectant. 
For  disinfecting  faeces  freshly  prepared  milk  of  lime  is  excellent.  It  is  made  by 
mixing  unslaked  lime  with  four  times  its  volume  of  water.  An  equal  quantity 
should  be  added  to  the  faeces  to  be  disinfected. 

Chlorinated  Lime. — This  can  be  purchased  in  air-tight  containers  and  when  the 
package  is  opened  it  should  give  off  a  powerful  oder  of  chlorine. 


APPENDIX  477 

For  a  working  disinfectant  solution  add  i  pound  to  4  gallons  of  water.  This 
is  satisfactory  for  mopping  floors  and  for  disinfecting  faeces,  sputum  and  urine, 
equal  parts  of  the  excreta  and  disinfecting  solution  being  mixed  and  allowed  to  stand 
for  one  hour.  For  disinfection  of  drinking  water  one  teaspoonful  of  chlorinated 
lime  to  i  pint  of  water  makes  a  stock  disinfectant.  For  use  one  teaspoonful  of 
this  stock  solution  is  added  to  .2  gallons  of  the  drinking  water  to  be  disinfected. 
Let  stand  at  least  one-half  hour. 

Eusol. — A  solution  containing  0.5%  hypochlorous  acid  and  known  as  eusol  has 
been  highly  recommended  in  the  treatment  of  gas  gangrene  wounds.  To  make  it 
put  27  grams  chlorinated  lime  (bleaching  powder)  in  a  Winchester  quart  flask  and 
cover  with  a  liter  of  water.  After  thorough  shaking  add  27  grams  of  boric  acid. 
After  shaking  the  mixture  should  stand  for  a  few  hours  and  then  be  filtered  through 
cotton  wool.  The  clear  solution  is  eusol.  It  must  be  kept  in  tightly  closed  bottles. 

INSECTICIDES 

The  following  notes  are  taken  chiefly  from  the  U.  S.  P.  H.  Service  directions. 

Sulphur  Dioxide — obtained  as  described  above — destroys  all  animal  life. 

In  the  case  of  vessels,  when  treated  for  yellow  fever  infection,  the  process  shall 
be  a  simultaneous  fumigation  with  sulphur  dioxide,  2%  volume  gas,  and  two  hours' 
exposure  in  order  to  insure  the  destruction  of  mosquitoes. 

In  the  case  of  vessels  when  treated  for  plague  the  process  with  sulphur  dioxide 
shall  be  as  follows: 

Without  cargo:  The  simultaneous  fumigation  with  sulphur  dioxide  gas  not  less 
than  2%  for  six  hours'  exposure. 

With  cargo:  Fumigation  with  sulphur  dioxide  gas,  4%,  six  to  twelve  hours' 
exposure,  according  to  stowing. 

Infected  vessels  may  require  partial  or  complete  discharge  of  cargo,  and  frac- 
tional fumigation  for  efficient  deratization. 

Pyrethrum. — The  fumes  of  burning  pyrethrum  may  be  used  to  destroy  mos- 
quitoes in  places  where  there  are  articles  liable  to  be  injured  by  the  use  of  sulphur. 

Four  pounds  per  1000  cubic  feet  space  for  two  hours'  exposure  will  kill  all,  or 
practically  all,  of  the  mosquitoes  but  precautions  should  be  taken  to  sweep  up  and 
destroy  any  that  may  have  escaped. 

Pyrethrum  stains  walls,  paper,  etc. 

The  Oxides  of  Carbon,  as  used  at  Hamburg,  are  efficient  to  destroy  rats  but  do 
not  kill  fleas  or  other  insects.  They  are  obtained  by  burning  carbon,  coke,  or  char- 
coal, in  special  apparatus,  and  the  gas  as  produced  consists  of  about  5%  carbon 
monoxide,  18%  carbon  dioxide,  and  77%  nitrogen. 

Twenty  kilos  of  carbon,  coke,  or  charcoal  are  used  for  every  1000  meters  of 
space.  The  gas  is  allowed  to  remain  in  the  ship  for  two  hours  and  from  seven  to 
eight  hours  are  allowed  for  it  to  leave  it.  This  is  about  equivalent  to  i  K  pounds 
of  carbon  (coke)  to  1000  cubic  feet  of  air  space.  As  this  gas  is  very  fatal  to  man  and 
gives  no  warning  of  its  presence,  being  odorless,  a  small  amount  of  sulphur  dioxide 
should  be  added  to  give  warning  of  its  presence.  As  it  does  not  kill  fleas  it  cannot 
be  depended  on  for  complete  work,  where  there  is  evidence  of  plague  among  rats 
on  the  vessel,  as  the  infected  fleas  would  infect  the  rats  coming  aboard  after  the 
deratization. 


478  APPENDIX 


The  articles  named  as  disinfectants  which  can  obviously  destroy  animal  life  can 
be  used  for  that  purpose  when  applicable,  as  steam  for  bedding,  fabrics,  etc.  For- 
maldehyde is  not  applicable  for  this  purpose. 

Pulicides. — For  fleas  the  best  insecticides  are  (i)  crude  petroleum  (fuel  oil)  which 
is  at  times  called  Pesterine,  (2)  an  emulsion  of  kerosene  oil  made  as  follows:  kerosene 
20  parts,  soft  soap  i  part  and  water  5  parts.  The  soap  is  dissolved  in  the  water  by 
aid  of  heat  and  the  kerosene  oil  gradually  stirred  into  the  hot  mixture. 

For  cockroaches  there  is  nothing  so  good  as  sodium  fluoride.  By  sprinkling  the 
powder  about  the  haunts  of  the  cockroaches  they  are  gotten  rid  of  in  a  few  days. 

Pediculicides . — Owing  to  their  great  importance  in  transmitting  typhus  fever 
and  relapsing  fever  the  destruction  of  human  lice  is  a  vital  consideration. 

While  the  body  louse  is  the  important  transmitting  agent,  the  head  louse  and  pos- 
sibly the  crab  louse  should  also  be  destroyed. 

The  subject  of  pediculosis  has  been  much  discussed  on  account  of  its  importance 
among  the  troops  in  the  European  war.  In  Shipley's  book  on  the  "Minor  Horrors 
of  War"  the  following  methods  of  destroying  lice  are  given.  Fernet  gives  the  follow- 
ing instructions  for  the  body  louse. 

1.  All  body  and  bed-linen  and  clothes  should  be  baked  or  sterilized  by  boiling. 

2.  Unguentum  staphisagriae  should  be  applied  to  neck-bands  of  vests  and  shirt  in 
the  region  of  the  neck. 

3.  Alkaline  baths  to  soothe  the  irritated  skin. 

Flowers  of  sulphur  sprinkled  in  the  bed  and  in  the  clothes  is  very  useful. 

Major  Lelean  recommends:  A  2-inch  loose-woven  bandage  is  made  into  a 
tubular  bag.  Into  this  bag  2  teaspoonsful  of  the  following  powder  is  poured  and 
evenly  distributed.: 

Naphthalene  96% 

lodoform  2% 

Creasote  2% 

The  bag  is  then  tied  round  the  waist,  and  it  is  said  that  all  lice  are  killed  within 
twenty-four  hours. 

Moor  head  advises  the  dusting  of  flowers  of  sulphur  on  the  clothes  but  many  re- 
ports indicate  the  inefficiency  of  this  method. 

For  head  lice  Fernet  recommends : 

1.  Prevention:  hair  to  be  kept  close  cropped  and  clean. 

2.  For  the  nits:  wipe  them  off  with  a  solution  of  i  in  30  carbolic  acid. 

3.  For  the  lice  themselves:  Unguentum  hydrargyri  ammoniat.  dil.  (gi.  xto  i  oz.), 
or  any  fatty,  sticky  body  well  rubbed  into  the  back  of  the  head.     Paraffin  lamp-oil 
(kerosene)  also  good,  but  not  to  be  used  near  a  naked  flame  or  light. 

Blanchard  considers  camphorated  alcohol  or  warm  vinegar  containing  i  to  1000 
of  corrosive  sublimate  as  useful  for  head  lice.  He  also  suggests  the  fumigation  of 
clothes  with  tobacco  as  valuable  for  body  lice. 

Castellani  and  Jackson  have  gone  most  extensively  into  the  matter  of  louse  des- 
truction. Their  conclusions  are  as  follows:  i.  In  regard  to  solid  and  liquid  insecti- 
cides, the  substances  which  have  been  found  to  be  deleterious  to  body-lice  are,  in 
the  order  of  their  efficiency:  Kerosene  oil,  vaseline,  guaiacol,  anise  preparations, 
iodoform,  lysol,  cyllin  and  similar  preparations,  carbolic  acid  solution,  naphthaline, 
camphor. 


ran 


APPENDIX  479 

Pyrethrum  has  a  very  feeble  action  on  lice,  while  boric  acid,  sulphur,  corrosive 
sublimate,  and  zinc  sulphate,  when  used  in  powder  form,  have  apparently  no  action 
whatever.  As  regards  bedbugs,  kerosene  oil  is  the  best  insecticide.  Next  to  it 
comes  guaiacol,  one  of  the  most  active  drugs  of  those  tried. 

2.  For  use  against  lice  on  a  large  scale,  as  among  troops  and  prisoners,  perhaps  the 
best  insecticide  powder  is  naphthaline.  This  substance  has  a  lower  licecide  action 
than  kerosene  oil,  guaiacol,  iodoform  and  anise  preparations,  but  it  has  less 
displeasing  odor  than  the  first  three  named.  In  stored  blankets  and  clothing 
it  is  also  practicable  and  of  use,  as  frequently  lice  are  found  upon  the  clothing 
and  blankets  stored  through  the  summer. 

Medical  Director  H.  G.  Beyer,  U.  S.  N.,  gives  the  following  description  of  the 
method  of  louse  destruction  employed  by  Lenz  at  the  prison  camp  at  Puchheim. 
On  the  arrival  of  a  large  transport  of  prisoners,  the  clothes  they  wear  on  their  persons 
are,  first  of  all,  subjected  to  steam-disinfection,  whereby  a  great  mass  of  lice,  includ- 
ing eggs,  are  destroyed.  The  hair  on  the  bodies  of  the  men  is  then  shorn  with  a 
machine,  and  they  are  lathered  over  with  soft  soap  and  put  into  a  bath.  The  now 
disinfected  clothes  are  put  on  again  and  treatment  with  naphthalin  is  begun  under 
the  supervision  of  the  sanitary  police.  Every  person,  at  bedtime,  has  a  handful  of 
finely  powdered  naphthalin  put  into  his  clothes,  introduced  through  the  opening 
at  the  neck.  He  is  made  to  sleep  that  night  with  all  his  clothes  on  him.  The  body 
heat  causes  the  naphthalin  to  evaporate,  the  vapors  killing  not  only  the  remaining 
lice,  but  also  most  of  the  remaining  eggs.  If  this  treatment  is  repeated  twice  more, 
regardless  of  whether  living  lice  are  found  or  not  at  the  time,  a  thorough  and  com- 
plete louse  disinfection  will  be  assured.  Lenz  does  not  mention  any  disagreeable 
effects  of  the  naphthalin  vapors  on  the  men  themselves.  When  dealing  with  trans- 
ports of  smaller  numbers  of  individuals,  Lenz  states  that  he  has  succeeded  even 
without  the  employment  of  steam-sterilization  of  the  clothes.  The  advantages 
of  the  naphthalin  method  as  used  by  him  are  given  as  follows: 

1.  That  it  is  cheaper  than  any  other  method,  its  cost  per  person  being  \y±  cents. 

2.  That  it  does  not  interfere  with  the  service  efficiency  of  the  men. 

3.  That  it  requires  neither  special  apparatus  nor  places. 

4.  That  it  does  not  injure  clothing. 

5.  That  it  is  absolutely  noninjurious  to  the  health  of  the  men. 

Raticides.— For  exterminating  rats  and  in  this  way  secondarily  the  rat-fleas,  be- 
sides the  ordinary  poisons  such  as  As,  P,  etc.  Rucker  has  recommended  a  poison 
composed  of  plaster  of  Paris,  6  parts,  pulverized  sugar  i  part  and  flour  2  parts.  This 
mixture  should  be  exposed  in  a  dry  place  in  open  dishes.  To  attract  the  rats  the 
edge  of  the  dish  may  be  smeared  with  the  oil  in  which  sardines  have  been  packed. 

Larvicides. — Wise  and  Minett  report  good  results  from  the  use  of  crude  carbolic 
acid  as  a  larvicide  for  mosquitoes.  They  added  about  i  teaspoonful  for  each  2 
cubic  feet  of  water  in  the  pool.  Of  course,  the  ordinary  method  for  destroying  mos- 
quito larvae  is  by  covering  the  surface  of  the  water  in  the  cistern  or  pool  with  a  layer 
of  petroleum. 

I— ANATOMICAL  AND  PHYSIOLOGICAL  NORMALS 

In  examinations  in  the  pathological  or  chemical  laboratory  the  following  may  be 
considered  approximately  as  normal  findings: 


480  APPENDIX 

A.  Anatomical  normals  of  adult  males.     Averages. 

Adrenals.     The  two  adrenals  together  weigh  about  %  ounce  (10  grams)  or 
grams  each. 

Brain.     Weight  50  ounces  (1400  grams). 

Heart.  Weight,  n  ounces  (310  grams).  Length,  5  inches  (125  mm.).  Breadth, 
3^  inches  (87  mm.).  Thickness,  wall,  left  ventricle,  ^  inch  (9  mm.),  right  ven- 
tricle, Ko  inch  (2.5  mm.).  Circumference,  mitral  orifice,  4%  inches  (10.5  cm.). 
Circumference,  tricuspid  orifice,  5  inches  (12.5  cm.).  Circumference,  aortic  orifice, 
3*4  inches  (8  cm.).  Circumference,  pulmonary  orifice,  3%  inches  (9  cm.). 

Intestines.  Small  intestine,  24  feet.  Large  intestine,  5  feet.  Duodenum,  10 
inches. 

Kidneys.  The  average  weight  of  both  kidneys  is  about  n  ounces  (310  grams). 
The  left  kidney  weighs  about  6  ounces  (160  grams),  and  the  right  about  5^  ounces 
(150  grams).  The  thickness  of  cortex  is  about  ^i  inch. 

Liver.  Weight  45  to  50  ounces  (1600  grams).  Greatest  transverse  diameter,  9 
inches  (25  cm.).  Greatest  antero-posterior  diameter,  4^  inches  (n  cm.). 

Lungs.  Combined  weight  39  ounces  (uoo  grams).  Right  lung  about  2  ounces 
heavier  than  left. 

Pancreas.  The  weight  of  this  organ,  like  the  spleen,  is  quite  variable.  An 
average  weight  would  be  about  2^  ounces  (70  grams). 

Spleen.  Weight  6  ounces.  (175  grams).  Length  5  inches  (12.5  cm.).  Breadth, 
3  inches  (7.5  cm.).  Thickness,  i%  inches  (3  cm.).' 

Testes.  The  left  testicle  is  slightly  larger  than  the  right  and  its  weight  is  about 
%  of  an  ounce  (20  to  24  grams). 

B.  Physiological  Normals. 

Blood. 

Specific  gravity  1041  to  1067 

Specific  gravity  (blood-serum)  1026  to  1032. 

Haemoglobin 14%. 

Serum  albumin 4.52%. 

Paraglobulin 3.10%. 

Fibrinogen 0.42%. 

Glucose 0.84  to  0.1%. 

Sodium  chloride 0.65%. 

Cholesterol 0.15%. 

Non-protein  N 25  to  35  mg.  in  100  grams  blood. 

Urea  N 1 2  to  23  mg.  in  100  grams  blood. 

Uric  acid i  to  2  mg.  in  100  grams  blood. 

Creatinin /.  . .  .    i  to    2  mg.  in  100  grams  blood. 

Cerebro-spinal  fluid. 

Specific  gravity 1007  to  1010. 

Urea o.oi  to  0.05%. 

Total  proteid 0.025%  (Albumin  not  present  normally). 

Pressure  of  fluid 5  to  7.5  mm.  mercury  or  60  to  100  mm 

water. 


.. 


APPENDIX  481 

Faeces. 

Amount  in  twenty-four  hours  on  ordinary  mixed  diet,  no  to  170  grams  (Solids 

25  to  45  grams). 

Amount  in  twenty-four  hours  on  vegetable  diet  up  to  350  grams  (Solids  75 

grams). 

Average  daily  output,  moist  faeces  (Hawk)   100  grams. 
Gastric  juice. 

One  hour  after  Ewald  breakfast. 

Quantity 40  to  50  c.c. 

Total  acidity 40  to  60  (0.15  to  0.22%). 

FreeHCl 20  to  60  (0.05  to  0.2%). 

Combined %  to  3  (o.oi  to  0.1%). 

Contents  of  fasting  stomach  (residuum)  20  to  50  c.c.  (Rehfuss  tube). 

Total  acidity  of  residuum 30. 

Free  HC1  acidity 19. 

Respiration. 

Composition  of  alveolar  air:  Oxygen,  14.5%;  Carbon  dioxide,  5.5%;  Nitrogen 

80%. 

Some  give  CO2  alveolar  content  as  from  3.7  to  5.5  volume  per  cent.,  or  CO2 

tension  of  alveolar  air  as  from  35  to  40  mm. 

Air  hunger  in  diabetes  or  chronic  nephritis  only  begins  when  CO2  tension  has 

fallen  to  20  or  25  mm. 
Urine.     Amount  (American  male)  1 200  c.c. 

Specific  gravity 1.015  to  1.025 

Urea 2.3%  (35  grams  in  twenty-four  hours)  about 

90%  of  total  nitrogen. 

Uric  acid 0.05%  (.75  grams  in  twenty-four  hours). 

Creatinine 0.07%  (i  gram  in  twenty-four  hours). 

Ammonia 0.04%  (.7  gram  in  twenty-four  hours). 

NaCl 1.1%  (!6-5  grams  in  twenty-four  hours). 


INDEX 


Abbe"   condenser,   5 
Abderhalden   technique,    195 

Bronfenbrenner's,  196 
Abscess,  bacteria  in,  420 
Acanthia  lectularia,  268,  344,  348,  379 

rotundata,  348 
Acarina,   334 
Acidosis,   218,   402,   456 

tests  for,   220,   456,  462 
Acartomyia,  376 
Acetone,  for  sections  (see  tissue),  445 

in  urine,  465 
Achromia,  223 
Acid-fast  bacteria,   90,   91 

staining,  41 
Acid  proofing,  13 
Actinomycosis  (see  Discomyces),  143, 

392,  420 
^Edinae,  374 
Agar,  egg,  27 

gelatin,  North,  28 

glucose,  27 

glycerine,  27 

nutrient,  26 

plating,  49 

Vedder's  starch,  27 
Agglutination,  macroscopical,  169 

microscopical,  168 
Ainhum,  438 
Air,    bacteriological   examination   of, 

.  159 

Albumin  in  urine,  457 
Albumin  in  sputum,  392 
Albumose  in  urine,  458 
Aldrichia,  373 
Alexin,  165 
Allergy,  194 

Alveolar  air  tension,   220,   456 
Ambard  index,  402 


Amboceptor,  164 

native  in  sheep  cells,  178 
Ammonia  in  urine,  462 
Amoebae,  252 
Amcebae,  of  intestines,  252 

of  mouth,  258 

staining  of,  46,  256 
Anaemia,  aplastic,  224 

infantum,  241 

pernicious,  236 

primary,  235 

secondary,  237 
Anaerobes,  78 

Buchner  method,  79 

combination  method,  80 

cultivation  of,  78 

Liborius  method,  79 

Noguchi  method,  36 

roll  culture,  50 

Tirozzi's  method,  78 

Vignal  method,  80 

Wright  method,  80 

Zinsser  method,  80 
Anaerobiasis,  55 
Analogy,  248 
Anaphylaxis,  192 
Anaphylactic  shock,  193 
Anaphylactine,  194 
Anaphylatoxin,  194,  196 
Anatomical  normals,  480 
Ancylostoma  duodenale,  313,  32 5,  411, 

422 

Anginas,  61,  387 
Anguillula,  314,  396 
Animal  inoculations,  55,  167,  176 
Animal   parasites,    general   classifica- 
tion, 243,  245 

key  to,  245 

mounting  of,  450 


483 


484 


INDEX 


Animal    parasites,    nomenclature     in, 
246 

preservation  of,  450 
Anisocytosis,  223 
Anlage,  248 
Anophelinae,  373 
Anthomyia,  326,  354,  361 
Anthrax,  75 

symptomatic,  77 

vaccination,  76 
Antiformin,  390 
Antigen,  173,  175,  178 

bacterial,  185 
Antitoxin,  105,  164 

botulism,  82,  124 
.  diphtheria,  102,  382,  384 

pyocyaneus,  129 

tetanus,  85 
Antivenins,  380 
Appendicitis  blood  count,  233 

organisms  in,  61 
Arachnoidea,  334 
Argas,  340 

Arneth  index,  227,  228 
Ascaris,  canis,  331 

lumbricoides,  330 
Ascitic  fluid  (cytodiagnosis  in),  424 
Aspergillus,  concentricus,  142 

flavus,  142 

fumigatus,  142 

nidulans,  143 

pictor,  143 

repens,  142 

Auchmeromyia  luteola,  357 
Autopsy  culture  methods,  56 
Azolitmin,  29 

Babesia,  291 

Bacillus,  acidi  lactici,  128,  157 

acidophilus,  128 

acnes,  421 

aerogenes  capsulat.,  81,  87 

Aertyrck,  123,  433 

anthracis,  75 

anthracis  symptomat.,  77 

anthracoides,  77 

bifidus,  128 


Bacillus,  botulinus,  82,  124 
bulgaricus,  128,  157,  416 
chauvoei,  77 
cloacae,  128 
coli,  127,  151,  153 
coli  anaerogenes,  154 
coli,  in  water,  152 
diphtherias,  102 
dysenteriae,  125 
enteritidis  (Gartner),  124 
enteritidis  sporogenes,  87,  149 
fecalis  alkaligines,  119,  154 
fusiformis,  387 
icteroides,  119 
influenzas,  in 
lactis  asrogenes,  no,  127 
leprae,  97 
mallei,  101 
mycoides,  73,  74 
of  avian  tuberculosis,  93,  94 
of  Bordet-Gengou,  113,  438 
of  bovine  tuberculosis,  93 
of  chancroid,  113 
of  chicken  cholera,  114 
of  Danysz  virus,  117,  124 
of  Hofmann,  107 
of  hog  cholera,  114,  433 
of  Koch-Weeks,  in 
of  malignant  oedema,  81,  82 
of  Morax,  112 
of  smegma,  90,  96 
of  timothy  grass,  91 
of  trachoma  (Muller),  109 
of  Zur  Nedden,  113 
paratyphosus  (A.  and  B.),  123 
perfringens,  88 
pestis,  114 

phlegmonis   emphysematosae,  75 
pneumonias  (Friedlander)  ,110,114 
prodigiosus,  130 
proteus,  124 
pseudotuberculosis    rodentium, 

116 

psittacosis,  119 
pyocyaneus,  129 
subtilis,  73 
tetani,  84 


INDEX 


485 


Bacillus,  termo,  125,  395 

tuberculosis,  92 

tularense,  383 

typhi-exanthematici,  438 

typhosus,  119 

violaceus,  129 

vulgatus,  73 

xerosis,  108 

zopfii,  no 

Bacteria,   identification   of,   48 
Bacteriaemia,  414 
Bacteriuria,  401 
Balantidium  coli,  273 
Banti's  disease,  240 
Bartonella,  440 
Bed-bug,  in  Kala  azar,  268 
Belascaris,  331 
Bence-Jones  albumin,   458 
Benedict  sugar  test,  460 
Beriberi,  438 
Besredka  medium,  24 
Bienstock  group,  no 
Bile  acids  in  urine,  466 
Bile  media,  31 
Bile  pigments,  408,  417,  466 
Bilharziasis,  300 

infection  in,  303 
Binucleata,  250 

Blackwater  fever,  292,  401,  439 
Blastocystis  hominis,  2  72 
Blood,  chemical  examination  of,  454 
Blood  coagulation  rate,  215 

color  index  of,  222 

counting  red  cells,  203 

counting  white  cells,  204 

counting  with  microscopic  field, 
2.05 

cultures  of,  413 

differential  count  (normal),  228 

differential  count  (in  haemacytom- 
eter),  206 

dried  films,  208 

fixation  of,  210 

fresh  preparations,  206 

haemoglobin  in,  200 

making  preparations,  208 

non-protein  nitrogen  in,  455 


Blood,  normal  count,  222 

occult,  216 

red  cells  of,  222 

specific  gravity  of,  215 

spectroscopic  test,  218,  394 

staining  of,  211 

thick  films  of,  210 

tubercle  bacilli  in,  415 

tubes  for  collection,  18 

urea  in,  455 

viscosity  of,  214 

white  cells  of,  224 
Blood  platelets,  230 
Blood  serum,  coagulating  apparatus, 

12 

preparation  of,  30 
Blood  sugar,  454 
Boas-Oppler  bacillus,  416 
Bodo,  272 
Booker  group,  no 

Bordet  and  Gengou  bacillus,  113,  438 
Bordet  and  Gengou  phenomenon,  172 
Bothriocephalus,  309 
Bottle  bacillus,  421 
Botulism,  83 
Bouillon,  glycerine,  25 

calcium  carbonate,  25 

egg,  25 

Liebig's  extract  in,  23 

nutrient,  20 

standardizing  reaction  of,  22 

sterilization  of,  23 

sugar,  24 

sugar-free,  23 
Bovine  tuberculosis,  93 
Bronfenbrenner's  anaerobic  culture,  36 
Broth  media,  20 
Buccal  secretions,  386 
Burker's  haemacytometer,  202 

Calliphora  vomitoria,  357 
Calmette  reaction  in  Tb.,  95 
Capillary  pipettes,  17 

standardizing  of,  181 
Capsule  staining,  43 
Carbol-fuchsin  stain,  41 
Carriers,  in  cholera,  133 


486 


INDEX 


Carriers,  in  diphtheria,  102,  108,  384 

in  dysentery,  255 

in  typhoid  fever,  122 
Casts  in  urine,  399 
Cellia,  373 

Cells,  in  cytodiagnosis,  424 
Centrifuge,  16 
Cercomonas,  272 
Cerebrospinal  fluid,  425 

puncture  for,  425 

Wassermann  of,  173,  428 
Cestoda,  294,  303 

key  to  genera,  305 
Charcot-Leyden  crystals,  389,  405 
Chinosol,  476 

Chlorides  in  urine,  403,  466 
Chlorinated  lime,  476 
Chlorosis,  235 
Cholecystitis,  418 
Cholera,  131 

carriers  in,  133 

diagnosis,  135 

in  water,  155 

media  for,  34 
Cholera  red,  25,  135 
Chironomidae,  363 
Chlamydozoa,  293,  434 
Cholesterinized  antigen,  173 
Chromatin  stains,  212 
Chromidia,  250,  258 
Chromogens,  129 
Chrysomyia  macellaria,  357,  359 
Chrysops,  353 
Chyluria,  318,  396 
Citellus,  351 
Cladorchis  watsoni,  298 
Cladothrix,  136 
Classification,  animal  kingdom,  244 

arachnoidea,  334 

bacilli,  branching,  90 

bacilli,  gram  negative,  109 

bacilli,  spore  bearing,  73 

bacteria,  51 

cocci,  57 

filarial  worms,  320 

flat  worms,  294 

fungi,  136 


Classification,  insects,  344 

mosquitoes,  373 

myiasis  larvae,  361 

protozoa,  249 

round  worms,  313 

spirilla,  131 
Cleaning  fluid,  10 
Clonorchis  endemicus,  297 

sinensis,  297 
Coarctate  pupa,  354 
Cocci diaria,  282 
Coccidium  (see  Eimeria  and  Isospora), 

283 

Cockroach,  destruction  of,  478 
Coley's  fluid,  130 
Colloidal  gold  test,  426 
Colon  bacillus,  127,  153,  154 

in  water,  152 
Colonies,  isolation  of,  50 
Color  index,  222 
Colubrine  snakes,  377,  379 
Commensalism,  246 
Common  cold,  435 
Complement,  165,  172,  183,  185 

absorption  of,  172 

deviation  of,  171 
Complement  fixation,  bacterial,  185 
Conjunctival  infections,  381 
Conorhinus,  348 
Conradi-Drigalski  medium,  34 
Conradi-brilliant  green  medium,  34 
Copper  sulphate,  476 
Corrosive  sublimate,  476 
Coryza,  435 
Cover-glasses,  3 

Cover-glass  preparations,  38,  208 
Crithidia,  269 
Cryptococcus  gilchristi,  139 

linguae  pilosae,  139 
Ctenopsylla  musculi,  350,  351 
Culicinae,  374 
Culture  media,  agar,  26 

autolyzed  tissue,  35 

Besredka's,  24 

bile  media,  31 

blood  agar,  31 

blood  serum,  30 


INDEX 


487 


Culture  media,  bouillon,  20 

cholera  media,  34 

Dorsett's  egg  medium,  30 

egg  media,  30 

faeces  media,  33 

gelatin,  28 

gelatin  agar  (North),  28 

Hiss'  serum  water,  25 

litmus  milk,  28 

milk  agar,  31 

Noguchi  media,  36 

peptone  solution,  25 

Petroff's  Tb.  medium,  32 

potato,  29 

protozoal,  35 

roll,  36 

Russell's  double  sugar,  35 

starch  medium  of  Vedder,  27 

sterilization  of,  8 

sugar  bouillon,  24 

titration  of,  21 
Cycloleppteron,  373 
Cysticercus,  305 
Cystitis,  401 
Cytodiagnosis,  423 
Cytorrhyctes  luis,  293 

scarlatinas,  293 

vaccines,  293,  433 

Dark  ground  illumination,  6 
Davainea  madagascariensis,  306,  309 
Demodex  folliculorum,  327 
Deneke's  spirillum,  131 
Dengue,  439 

Dermacentor  andersoni,  342,  437 
Dermatobia  cyaniventris,  358,  359 
Desk-microscopic,  12 
Dhobies  itch,  142,  145 
Diazo  reaction,  465 
Dibothriocephalus  latus,  309 
Dicroccelium  lanceatum,  297 
Dieudonne's  cholera  medium,  34 
Differential    leukocyte     count,     228, 

229 
Diphtheria,  102 

carriers,  102 

diagnosis  of,  106 


Diphtheria,  diphtheria-like  bacilli,  107 

media  for  growing,  30 

Neisser's  stain,  42 

Schick  reaction,  106 

toxin  of,  104 
Diphtheroids,  108 
Diplococcus,  crassus,  58 

intracellular,  meningitidis,  68 

lanceolat.,  64 

Diplognoporus  grandis,  310 
Diptera,  351 

Dipylidium  caninum,  309 
Disinfecting  solution,  13,  476 
Disinfectants,  472 

solutions  for  laboratory,  13 
Distomiasis,  297 
Dorset's  egg  medium,  30 
Double  boiler,  12,  19 
Dracunculus,  315 
Dum  dum  fever,  267 
Dunham's  solution,  25 
Duodenal  fluid,  417,  472 

examination  of  472 

tests  for  ferments  in,  472 
Dysentery,  amoebae  in,  252 

bacilli,  125 

bacilli  in  faeces,  409 

Ear  affections,  385 

Eberth  group,  119 

Echinococcus  cysts,  310 

Echinorhynchus  gigas,  332 

Echinostoma,  299 

Edestin  test,  471 

Egg  broth,  25 

Ehrlich,  blood  film  method,  208 

granule  staining,  213,  227 

tri-acid  stain,  211 
Eimeria  stiedae,  283 
Emery's  test,  179 
Ekiri,  127 

Endodermophyton,  141 
Endo  medium,  33 
Endomyces  albicans,  139 
Endothelial     cell     in     cytodiagnosis, 

424 
Entamceba,  buccalis,  258 


INDEX 


Entamoeba,  coli,  252 

gingivalis,  258 

histolytica,  252 

tetragena,  252 
Eosinophiles,  227,  231 
Eosinophilia,  231 
Epidermophyton,  142 
Esbach  test,  458 
Escherich  group,  119 
Esmarch  roll  cultures,  50 
Espundia,  270,  422 
Eusol,  477 

Eustrongylus  gigas,  324 
Exudates,  423 
Eye-piece  (see  Ocular),  2 
Eye-strain,  4 
Eye  infections,  381 

Faeces,  405 

amoebae  in,  408 

bile  in,  408 

culturing,  33,  409 

diet  for  examination  of,  406 

fats  in,  407 

fermentation  test,  408 

pancreatic  test,  410 

plating  media,  33 

soaps  in,  407 
Fasciola  gigantea,  300 

hepatica,  297 
Fascioletta  ilocana,  299 
Fasciolopsis  buski,  298 
Fat  in  faeces,  407 
Fauces,  386 
Favus,  141 

Fehling  sugar  test,  459 
Fermentation  tubes,  1 1 
Ferments  in  duodenal  fluid,  417,  472 
Films  (blood),  208,  210 
Filter  pump,  16 
Filterable  viruses,  433 
Filaria,  bancrofti,  317 

demarquayi,  319 

embryos,  key  to,  319 

loa,  317 

medinensis,  315 

ozzardi,  319 


Filaria,  perstans,  318 

philippinensis,  318,  319 

powelli,  319 

volvulus,  319 
Fixation,  blood  films,  210 

tissues,  443 
Flagella  staining,  44 
Flagellata,  259 
Flat  worms,  294 
Fleas,  348 

key,  349 

Flugge's  droplet  infection,  93,  118 
Flukes,  294 

of  blood,  300 

of  intestines,  298 

of  liver,  297 

of  lungs,  299 
Focus,  microscopical,  3 
Fontana  spirochaete  stain,  47 
Foot  and  mouth  disease,  436 
Formalin,  443,  474 
Foulis'  cells,  424 
Friedlander  group,  no,  114 
Frozen  sections,  450 
Fungi,  Achorion,  141 

Ascomycetes,  138 

Aspergillus,  142 

classification  of,  136 

Cryptococcus,  139 

cultivation  of,  147 

diagnosis  of,  147 

Discomyces  bovis,  143 

Discomyces  madurae,  144 

Hyphomycetes,  143 

Inperfecti,  143 

Madurella  mycetomi,  145 

Malassezia  furfur,  145 

Microsporoides,  145 

Microsporum  audouini,  141 

Monilia,  145    , 

Mucor,  138 

Penicillium,  143 

Rhizopus,  138 

Saccharomycetes,  139 

Sterigmatocystis,  143 

Trichophyton,  140 

Trichosporum  giganteum,  146 


INDEX 


489 


Gall  stones,  410 
Gametes,  285 
Gartner  group,  123 
Gas  bacillus,  81,  87 
Gas  gangrene,  81 
Gas  production,  54 
Gastric  contents,  416,  469 

chemical  examination  of,  469 
Gastric  ulcer  streptococci,  61 
Gastrodiscus  hominis,  298 
Gelatin,  28 

liquefaction  of,  54 
General  paralysis  (spinal  fluid  in),  174, 

426 

Gentian  violet  stains,  39 
Giemsa's  stain,  45,  213 
Glanders,  101 

Glassware,  cleaning  of,  9,  427 
Globulin  tests,  427 
Glossina  palpalis,  355 
Gluzinski  test,  469 
Gnathostoma  siamense,  314,  422 
Gonococcus,  67 
Gonorrhoea,  67 
Goundou,  440 
Grabhamia,  376 
Gram  method,  39 

negative  bacteria,  40 

positive  bacteria,  40 

solution,  39 

Granular  degeneration  (red  cells),  223 
Granules  (white  cells), 226 
Guinea  worm,  315 

Haemacytometer,  202 
Haemadipsa  ceylonica,  333 
Hsematopota,  353 
Haematoxylin  stain,  46,  213,  449 
Haematuria,  401 
Haemin  crystals,  217 
Haemoglobin  estimation,  200 
Haemoglobinometers,  Miescher's,  200 

Sahli's,  200 

Tallquist,  201 

Haemoglobinophilic  bacteria,  in,  112 
Haemoglobinuria,  216,  401 
Haemolysin  production,  176 


Haemolysis  tests,  176 
Haemosporidia,  283 
Haffkine,  cholera  vaccine,  134 

plague  prophylactic,  134 
Halzoun,  297 
Hanging  drop,  n 
Hayem's  blood  diluent,  203 
Hemokonia,  230 
Heredity,  247 
Herpetomonas,  269 
Heterogenesis,  248 
Heterophyes  heterophyes,  299 
Hirudo,  medicinalis,  332 

nilotica,  332 
Hiss'  serum-water,  25 
Histoplasma,  271 
Hodgkin's  disease,  239 
Hog  cholera  virus,  433 
Homology,  248 
Hook  worms,  325 
Hoplopsyllus,  349 
Hosts,  247 
Hydatid  disease,  310 
Hydrocele  agar,  31 
Hydrogen  dioxide,  476 
Hymenolepis,  nana,  307 

diminuta,  309 
Hyphomycetes,  143 
Hypoderma  diana,  358 

Illumination,  dark  ground,  6 
Immersion  objectives,  3 
Immune  sera,  antimicrobic,  164 

antitoxic,  163 

diphtheria,  105 

in  diagnosis,  166 

preparation,  166 

tetanus,  85 
Immunity,  active,  163 

natural,  161 

passive,  163 

Inactivation  of  serum,  165 
Incubators,  body  temperature,  15 

electrical,  15 

petroleum  lamp,  15 

room  temperature,  15 
Indican  in  urine,  465 


4QO 


INDEX 


Indol,  test  for,  25 
Influenza,  in 
Infusoria,  273 

Inoculation    animals     (tuberculosis) , 
48,  91 

animals  (plague),  116 

of  media,  49 
Insecticides,  477 
Inspissators,  12 
Insecta,  344 
Intestinal  amoebae,  252 

flagellates,  271 

myiases,  360 
lodophilia,  214 
Isospora  bigemina,  283 
Itch  mite,  337 
Ixodidae,  338 

Japanese  river  fever,  336,  442 
Joints,  gonococcus  in,  68 

Kaiserling  solution,  452 

Kala  azar,  267 

Kedani  mite,  336,  442 

Key  to  branching,  curving  bacilli,  90 

to  cocci,  57 

to  filarial  embryos,  320 

to  fleas,  349 

to  Gram-negative  bacilli,  109 

larvae  in  myiases,  361 

to  spirilla,  131 

to  spore-bearing  bacilli,  73 
Kidney  diseases,  table,  404 
Koch's  postulates,  55 
Kundrats  lymphosarcoma,  240 

Laboratory  desks,  13 
Lactic-acid  bacteria,  128,  156 
Lactophenol,  451 
Lamblia  intestinalis,  273 
Lamp,  primus,  18 
Lange's  colloidal  gold  test,  426 
Large  mononuclear  increase,  234 
Larvae,  fly,  361 

key  to  dipterous,  361 

mosquito,  368 

mounting,  451 


Leeches,  332 
Leishmania,  267 

donovani,  267 

infantum,  268 

media  for,  35 

tropica,  269 
Leishmaniases,  267 
Leprosy,  97 

cultural  questions,  98 

diagnosis  of  83,  100 

in  rats,  99 
Leptomonas,  269 
Leptothrix,  136 
Leukaemia,  238 

lymphatic,  239 

splenomyelogenous,  238 
Leukocytosis,  232 
Leukopenia,  231 
Levaditi  stain,  447 
Leydenia  gemmipara,  249 
Lice,  345,  478 

destruction  of,  478 
Light  in  microscopical  work,  5 
Linguatula  rhinaria,  343 
Liquefaction  of  gelatine,  54 
Litmus,  29 
Liver  abscess,  420 
Loeffler  serum,  30 
Loemopsylla  cheopis,  350 
Luetin,  262 
Lumbar  puncture,  425 
Lymphocytes,  large,  225 

small,  225 

Lymphocytosis,  234 
Lymphosarcoma,  239 
Lyon's  blood  tube,  18 

Madura  foot,  144 
Macrogamete,  285 
Magnifying  power,  199 

of  oculars,  2 
Malaria,  284 

cultivation,  289 

diagnosis  of,  286 

differential  tables,  290 

index,  286 

life  cycle,  284 


INDEX 


491 


Malaria,  life  history,  284 

Romano wsky  stain  in,  291 
Mallein,  102 

Mallory's  amoeba  stain,  46 
Malta  fever,  71 
Mansonia,  376 
Marchi  method,  449 
Mast  cells,  227 
Measles,  436 
Megarhininae,  373 
Meat  poisoning,  83,  123 

group  of  bacteria,  123 

toxin  of,  83 
Malignant  pustule,  75 
Mechanical  stage,  I 
Media  (see  culture  media),  19 
Megaloblast,  224 
Megakaryocytes,  230 
Melaniferous  leukocytes,  235 
Meningococcus,  68 
Metorchis  truncatus,  298 
Mett  test  for  pepsin,  471 
Micrococcus,  62 

catarrhalis,  70 

cinereus,  58 

melitensis,  71 

pharyngis  siccus,  58 

rheumaticus,  61,  435 

tetragenus,  62 
Microgametocyte,  285 
Micrometer  disk,  197 

standardization  of,  198 

screw,  i 

Micro  metry,  197 
Microscope,  i 

care  of,  2 

dissecting,  i 
Microscopical  sections  (see  tissue),  443 

quick  diagnostic  method,  446 
Milk,  bacteriological  examination  of, 

155 

B.  bulgaricus  in,  157 

lactic-acid  bacteria  in,  157 

leukocytes  in,  157 

pasteurization  of,  158 
Minimal  lethal  dose,  105 
Mites,  335 


Mollusc  hosts  in  schistosomiasis,  303 
Monilia  albicans,  145,  441 

Candida,  146 

in  sprue,  442 

Mononuclear,  leukocytes,  225 
Mosquitoes,  anatomy  of,  365 

classification  of,  372 

dissection  of,  370 

larvae  of,  368 

ova  of,  367 

pupae  of,  369 

transmitters  of  malaria,  373 
Motility,  53 

Brownian,  51 

current,  51-53 
Moulds  (see  Fungi),  136 
Mounting  parasites,  450 
Much's  granules,  41 
Mucidus,  376 
Mumps,  437 
Mus  norvegicus,  351 
Musca  domestica,  353 
Muscidae,  353 
Mutualism,  245 
Myeloblasts,  230 
Myelocytes,  229 
Myiases,  359 

key  to  larvae  in,  361 
Myzomyia,  373 
Myzorhynchus,  373 

Nasal  infections,  diphtheria  in,  384 

leprosy  in,  384 
Nastin  in  leprosy,  99 
Necator  americanus,  325 
Negri  bodies,  430 
Neisser's  stain,  42 
Nematocera,  352 
Nematoda,  313 
Neosporidia,  282 
Nervous  tissue,  449 
Nissl  method,  449 
Nitrogen  determination,  455-462 
Nocardia,  147 
Noguchi  test,  174 

media  for  treponemata,  36 


492 


INDEX 


Nomenclature,  in  animal  parasitology, 

246 

law  of  priority  in,  246 
Nonprotein  nitrogen,  455 
Normal  solutions,  452 
Normoblasts,  223 
North's  gelatin  agar,  28 
Novy  MacNeal  (N.N.N.)  medium,  35 
Nucleo-protein  test,  458 
Numerical  aperture,  4 
Nylander  sugar  test,  461 
Nyssorhynchus,  373 

Objectives,  2 
Occult  blood,  216 
Ocular  infections,  381 

animal  parasites  in,  381 

bacilli  in,  382 

gonococcus  in,  382 

M.  catarrhalis-  in,  383 

pneumococcus  in,  382 
Oculars,  2 

CEsophagostoma  brumpti,  325 
CEstridae,  358 
Oidium,  145 

Onchocerca  volvulus,  319 
Opisthorchis,  felineus,  298 

noverca,  298 

sinensis,  297 
Opsonic  power,  1 86 

apparatus  in,  188 

determination  of,  188 
Ornithodoros,  340 
Oroya  fever,  440 
Orthorrhapha,  351 
Otitis,  385 
Ova  in  faeces,  411 
Oxyuris  vermicularis,  331 

Pancreatic  tests,  410,  472 
Pandy  globulin  test,  428 
Pangonia,  353 
Panoptic  staining,  46,  448 
Paragonirhus  westermani,  299 
Paraplasma  flavigenum,  292,  442 
Parasitism,  243 
Parthenogenesis,  248 


Pasteur  elloses,  114 
Pasteurized  milk,  158 
Pasteur  treatment,  429 
Pathological  sections,  443 
Pebrine,  282 
Pediculicides,  478 
Pediculoides  ventricosus,  337 
Pediculus  capitis,  345 

vestimenti,  345 
Pellagra,  440 

Penicillium  crustaceum,  142 
Pentastomida,  342 
Pepsin  tests,  471 
Petri  dishes,  9,  48 
PetrofFs  T  b.  medium,  32 
Pfeiffer's  phenomenon,  134 
Phagocytosis,  186 
Pharyngeal  secretions,  386 
Phenol  coefficient,  473 
Phenolphthalin  test,  217 
Phenolsulphonephthalein  test,  467 
Phenylhydrazin  test,  459 
Phlebotomus,  364 
Phthirius  pubis,  346 
Physaloptera,  325 
Physiological  normals,  480 
Piedra,  146 
Pinta,  143 
Pipettes,  bacteriological,  18 

capillary  bulb,  17 
Piroplasmata,  291 
Pirquet,  von,  reaction  in  T  b.,  96 
Plague,  114 

diagnosis  of,  116 

flea  in,  117,  349 

pneumonia,  118 

prophylaxis,  118 
Plasmodium  vivax,  287 

falciparum,  288 

malarias,  288 
Platinum  wire,  14 

loops,  14 

Pleural  fluids  (cytodiagnosis),  423 
Pneumococcus,  64 
Poikilocytes,  223 
Poliomyelitis,  436 
Polymorphonuclear  leukocytes,  227 


INDEX 


493 


Porocephalus  constrictus,  343 
Precipitins,  166-170 
Protista,  248 
Protozoa,  250 

culture  of,  35 

discussion  of,  250 

staining  of,  45 
Prowazekia,  272 
Pseudoleukaemia,  239 
Psychodidae,  364 
Pulex,  cheopis,  350 

irritans,  350 
Pulicidae,  348 
Pupipara,  352 
Pus,  cultures  from,  419 

in  urine,  401 

tetanus  in,  86 
Pyorrhoea,  amoebae  in,  258 
Pyosis,  63 
Pyretophorus,  373 

Rabies,  429 

preservation  of  dog  in,  431 

Rats,  351 

Rat-bite  disease,  441 

Reaction  of  media,  21,  51 
standardization  of,  21 

Reagine  of  syphilis,  173 

Red  blood-cells,  counting  of,  203 
normal,  222 
nucleated  red  cells,  223 
polychromatophilia,  223 
punctate  basophilia,  223 

Relapsing  fever,  260 

Renal  efficiency  determinations,  402 

Rhabditis  pellio,  314 

Rheumatism  (acute),  435 

Rhinosporidium,  292 

Rhizoglyphus  parasiticus,  337 

Rhizopoda,  251 

Rhizopus,  138 

Rhynchota,  346 

Rice  cooker,  12,  19 

Ring- worms,  140 

Rocky  Mountain  spotted  fever,  437 

Roetheln,  437 

Romano wsky  stains,  212  . 


Room  temperature  incubators,  15 

Ross  thick  film,  210 

Round  worms,  313 

Row's  haemoglobin  medium,  36 

Russell's  double  sugar  medium,  35 

Sabouraud's  medium  for  moulds,  148 
Saccharomyces,  anginosse,  139 

blanchardi,  139 

cerevisiae,  139 
Salt  retention,  403 
Sarcina  lutea,  62 
Sarcophaga  carnaria,  357-361 
Sarcopsylla  penetrans,  351 
Sarcoptes  scabiei,  337 
Sarcosporidia,  292 
Scarlet  fever,  61,  437 
Schick  reaction,  106 
Schilling-Torgau  differential  count, 

229 
Schistosomum  haematobium,  300 

japonicum,  302 

mansoni,  301 
Schizotrypanum,  266 
Screw  worm,  359 

Sections,  making  and  staining,  443 
Sensitization,  165 
Sensitized  vaccines,  192 
Septicaemia,  414 

Serum  (see  immune  serum),  163-165 
Sewage,  in  water,  149 
Sheep  cells,  178 
Shiga's  bacillus,  125 
Simulidae,  363 
Siphonaptera,  348 
Siphunculata,  345 
Skin  infections,  42 1 

itch  mite,  337,  422 

leprosy  in,  421 

pus  cocci  in,  421 

sarcopsylla  in,  422 
Sleeping  sickness,  264 
Slides,  cleaning,  10 

concave,  n 
Smallpox,  432 
Snakes,  377 
Sparganum,  mansoni,  312 


494 


INDEX 


Sparganum,  prolifer,  312 
Spectroscope,  218,  393 
Spinal  fluid,  173,  425 
Spirillum  cholerae  asiaticae,  131 

metschnikovi,  131 

of  Finkler-Prior,  131 

tyrogenum,  131 
Spirochaeta,  259 

duttoni,  260 

recurrentis,  260 

refringens,  261 

vincenti,  261,  386 
Spirochaete  staining,  47 
Spiroschaudinnia,  260 
Splenic  anaemia,  241 
Splenomegaly,  240 
Spores,  spore-bearing  bacilli,  73 

staining,  45 
Sporotrichosis,  146 
Sporotrichum  beurmanni,  146 
Sporozoa,  282 
Sprue,  441 
Sputum,  389 

albumin  test  in,  392 

amoebae  in,  392 

antiformin  for,  390 

centrifugalization  for  T.  B.,  390 

culturing,  32,  391 

fixing  smears,  391 

Paragonimus  eggs  in,  392 

Petroff 's  T  b.  culturing,  32 

plague  pneumonia,  392 
Stage,  warm,  5 
Staining  methods,  38 
Stains,  acid  fast,  41 

agar  jelly,  45 

Archibald's,  42 

Balch's,  212 

capsule,  43 

carbol  fuchsin,  39 

carmine  for  worms,  45 1 

flagella,  44 

Fontana  spirochaete,  47 

for  Negri  bodies,  431 

Giemsa's,  213 

Gram's  method,  39 

haematoxylin,  46,  213,  449 


Stains,  Herman's,  41 

iron  haematoxylin,  46,  447 

Leishman's,  212 

Levaditi's,  447 

Loffler's  methylene  blue,  39 

Mallory's,  46,  47,  449 

Neisser's,  42,  107 

Nicolle's,  447 

panoptic,  46,  448 

Pappenheim's,  42 

Ponder's  diphtheria,  43 

protozoal,  45 

Romano wsky,  212 

Rosenbusch's,  46 

Smith's  formol  fuchsin,  41 

spore,  45 

tri-acid,  211 

Van  Giesen's,  447 

Wright's,  212 
Staphylococcus,  63 

epiderrriidis  albus,  63,  421 

pyogenes  albus,  63 

pyogenes  aureus,  63 
Stegomyia,  374 
Sterigmatocystis,  143 
Sterilization,  Arnold,  7 

autoclave,  8 

glass  ware,  9 

hot  air,  6 

pathogenic  bacteria,  9 
Stomach  contents,  416,  469 

Boas-Oppler  bacillus,  416 

cancer  cells  in,  416 
Stomoxys,  355 
Stool  examination,  405 
Streptococcus,  58 

anaerobic,  414 

capsulatus,  65 

coli  gracilis,  57 

fecalis,  57 

haemolyticus,  60 

lacticus,  156 

mucosus,  65 

pyogenes,  60 

viridans,  60 
Strep  tothrix,  136 
Strong,  cholera  prophylactic,  134 


INDEX 


495 


Strong,  plague  vaccine,  118 
Strongylidae,  324 
Strongyloides  stercoralis,  314 
Strongylus  apri,  324 
Sugar  in  blood,  454 

in  urine,  459 

media,  24,  153 
Sulphur  disinfection,  475 
Swabs,  9 

Syphilis,   fixation  of  complement  in 
diagnosis,    173 

Giemsa's  stain  in,  261 

precipitate  tests  in,  185 

smears  in,  262 

Tabanus,  352 
Tables,  insects,  344 

mosquitoes,  373 

of  arachnoids,  334 

of  filarial  worms,  320 

of  flat  worms,  294 

of  fungi,  136 

of  myiasis  larvae,  361 

of  parasitic  animals,  245 

of  protozoa,  249 

of  round  worms,  313 

pressure  and  temperature,  9 

urinary  findings,  404 
Taenia,  africana,  307 

echinococcus,  310 

saginata,  306 

solium,  307 
Taeniorhynchus,  376 
Tape  worms,  adult,  303 

somatic  or  larval,  310 
Telosporidea,  282 
Terminology,  247 
Test-tubes,  9 
Tetanus,  84 

antitoxin,  85 

diagnosis  of,  81,  86 

toxin,  85 

Tetragena  nucleus,  253 
Tetramitus  mesnili,  272 
Thalman's  gonococcus  medium,  32 
Theobaldia,  376 
Thick  film  blood  smears,  210 


Thionin,  42,  211 
Thorn-headed  worm,  332 
Throat  examination,  386 
Thrush,  139,  145,  386 
Tick  fever,  260,  340 
Ticks,  338 

classification  of,  334 

life  history,  339 
Tinea,  140 

imbrica^a,  141,  142 

versicolor,  145 
Tissue,  acetone  method,  445 

chloroform  method,  445 

dehydration  of,  444 

fixation  of,  443 

imbedding,  444 

nervous,  449 

preparation  of  for  sections,  443 
Titration  of  media,  21 
Toisson's  solution,  203 
Tongue  worms,  342 
Toxascaris,  331 
Toxic  split  proteids,  194 
Toxin,  164 

diphtheria,  105 

tetanus,  85 

Toxone  of  diphtheria,  105 
Trachoma,  112,  437 

and  Koch- Weeks  bacilli,  112 
Transfusion  blood  tests,  216 
Transitional  leukocyte,  226 
Transudates,  423 
Trematoda,  294 
Treponema,  261 

culture  media  for,  36 

pallidum,  261 

pertenue,  263 
Trichinella  spiralis,  321 
Trichinosis,  321 
Trichocephalus  trichiurus,  321 
Trichomonas  intestinalis,  272 

vaginalis,  272,  395 
Trichophyton  mentagrophytes,  141 

Sabouraudi,  140 

tonsurans,  140 

Trichostrongylus  instabilis,  324    . 
Trichuris,  321 


496 


INDEX 


Triodontophorus  deminutus,  324 
Trombidiidae,  335 
Trombidium  holosericeum,  335 
Trypanocides,  264 
Trypanosoma,  brucei,  266 

equinum,  266 

equiperdum,  266 

evansi,  266 

gambiense,  264 

lewisi,  266 

rhodesiense,  264 
Trypanosomiasis,  264 
Tsetse  flies,  265,  355 
Tuberculin,  95 
Tuberculosis,  bacilli  of,  92 

atrium  of  infection,  93 

avian  and  fish,  94 

bovine  and  human,  93 

British  commission  report,  93 

Calmette  eye  reaction,  95 

diagnosis  of,  95 

in  guinea-pigs,  91 

intradermic  test,  96 

Much's  granules,  41 

secondary  infections,  94 

staining  methods,  41 

von  Pirquet  skin  reaction,  96 
Tuberculins,  95 
Tubes,  fermentation,  8 

test-tubes  for  agglutination,  169 

Wright's  U-tube,  17 
Turck,  irritation  cells,  229 

ruling  on  haemacytometer,  202 
Typhoid,  agglutination  in,  121 

and  water  supply,  123,  155 

blood  cultures,  121,  412 

carriers,  122 

dead  cultures,  169 

gall  stones  in,  122 

in  water,  155 

Mandelbaum's  thread  culture,  1 70 

relapses  in,  121 

serum  for,  121 

statistics,  123 

vaccination  in,  122 
Typhus  fever,  438 
Tyroglyphus  longior,  337 


Ultra  microscope,  6 
Urea,  393 

in  blood,  455 

in  urine,  463 

index  of  excretion,  402 
Urease  tests,  455,  464 
Uric  acid  tests,  466 
Urine,  393 

casts  in,  399 

chemical  examination,  456 

chylous,  396 

culturing  of,  395 

epithelium  in,  399 

moulds  in,  395 

reaction  of,  462 

schistosomum  eggs  in,  396 

sediment  in,  396 

smegma  bacillus  in,  395 

starch  grains  and  fibres  in,  400 

sugar  in,  459 
Urobilin,  466 
Urotropin  and  urine,  467 
Urticarial  fever,  302 
Uta,  422 

Vaccines,  189 

doses  of,  191 

preparation  of,  189 

sensitized,  192 

standardizing  of,  191 

typhoid  and  paratyphoid,  124 
Vaccinia,  432 
Vacuum  bottle,  80 
Varicella,  438 

Vedder's  starch  medium,  27 
Verruga  peruana,  442 
Vincent's  angina,  386 
Viperine  snakes,  379 
Vitamines,  439 
Voges-Proskauer,  25 

Warm  stages,  5 
Wassermann  test,  177 

Emery,  179 

Fleming,  184 

Noguchi,  174 

statistics,  184 


INDEX 


497 


Water,  bacteriological  examination, 
149 

cholera  spirillum  in,  155 

colon  bacillus  in,  152 

disinfecting,  477 

qualitative  bacteriological  exami- 
nation, 151 

quantitative    bacteriological    ex- 
amination, 150 

typhoid  bacillus  in,  155 
Weigert  method,  449 
Whip  worms,  321 
White  blood-cells,  224 

counting  of,  204 

normal  count,  228,  229 
Whooping  cough,  438 
Widal  tests,  168 
Wing  venation,  352-354 


Wollf  and  Junghan's  test,  470 

Woolsorter's  disease,  75 

Working  distance,  3 

Wright's  blood  stain,  212 

method  for  anaerobes,  80 
method   for    standardizing 
cines,  190 

Xenopsylla  cheopis,  350 
Xerosis  bacillus,  108 

Yaws,  263 
Yersin's  serum,  118 
Yellow  fever,  442 

Zettnow's  flagella  stain,  44 
Zur  Nedden's  bacillus,  113 


p 


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10* 

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Equivalent  Fahrenheit  and 
Centigrade  tables  for  the  temper- 
atures in  common  use  in  labora- 
tories: 


i.  Those  for  sterilization  of 
dressings  and  media.  Also  f for 
certain  disinfection  of  spore-bear- 
ing bacterial  contamination 
(autoclave  temperatures). 


P.  C. 


2.  Those  for  pasteurization 
and  sterilization  of  bacterial  vac- 
cines. Also  for  paraffin  bath 
(pasteurizing  temperatures). 


1*0 


57 


V04- 


1* 

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f 

— 

75 

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1 

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£ 

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32. 

3.  Those  for  growing  impor- 
tant pathogenic  organisms  (body 
temperatures). 


4.  Those  for  culturing  gelatin 
(melting  point,  25°C.)  as  in  water 
work  (room  temperatures). 


5.  Those  employed  in  preserv- 
ing biological  products  and  post- 
mortem material;  also  in  centri- 
fuging  experiments  to  separate 
complement  and  amboceptor 
(freezing  temperatures). 


160 


50  = 


320 


10   = 


10 


AMERICAN  STERILIZER co, 

ERJE.F*. 


UNITS  IN  COMMON  USE  IN  LABORATORIES 

Cubic  Meter. — Unit  of  space  for  the  number  of  organisms  in  air.  It 
contains  1000  liters.  It  is  equal  to  1.308  cubic  yards  or  35.316 
cubic  feet.  One  thousand  cubic  feet,  the  unit  of  space  in  disinfec- 
tion, is  equal  to  28.3+  cubic  meters.  One  cubic  decimeter  is  one 
liter  and  equals  0.908  quarts  dry  measure  or  1.0567  quarts  liquid 
measure. 

Liter. — Unit  of  space  for  normal  volumetric  solutions.  It  contains 
1000  cubic  centimeters.  It  is  equal  to  1.0567  quarts  or  33.84- 
ounces.  A  liter  of  distilled  water  weighs  i  kilogram.  One  U.  S. 
gallon  is  equal  to  3785  c.c.  and  one  imperial  gallon  to  4543  c.c. 
One  fluid  ounce  equals  29.57  c.c. 

Cubic  Centimeter. — Unit  of  space  for  organisms  in  water,  milk,  vac- 
cines, etc.,  i  c.c.  =  0.27  fl.  dr.  There  are,  approximately,  20  drops 
in  i  c.c.  of  water,  provided  the  capillary  pipette  has  a  bore  of  about 
i  mm.  and  is  held  horizontally.  A  finely  drawn  capillary  pipette, 
held  vertically,  will  deliver  about  50  drops  from  i  c.c. 

Cubic  Millimeter. — Unit  of  space  for  blood-cells.  There  are  1000 
cubic  millimeters  in  i  cubic  centimeter  and  i  million  cubic  milli- 
meters in  i  liter.  In  water  analysis,  as  there  are  i  million  milli- 
grams in  one  liter,  parts  in  the  million  and  milligrams  per  liter 
are  the  same. 

i  Meter  =  39. 3  7  inches. 

i  Centimeter  =  0.393  7  inch.     Approximately,  2/5  inch. 

i  Millimeter  =  0.0393  inch.     Approximately,  1/25  inch. 

i  Inch  =  2 5. 4  mm. 

i  Yard  =  0.9 144  m. 

i  Kilogram  =  2. 2+  pounds  av. 

i  Centigram  =  0.154  grain. 

i  Milligram  =  0.01 54  grain.     Approximately,  1/64  grain. 

A  pound  avoirdupois  is  equal  to  453.59  gm. 

i  Oz.  avoirdupois  is  equal  to  28.35  gm. 

One  hundred  cubic  centimeters  of  a  saturated  solution  contains: 

Water  Alcohol 

Methylene  blue,  6.68  0.66  gram. 

Gentian  violet,  1.75  4.42  grams. 

Basic  fuchsin,  0.66  2.92  grams. 

Key  to  Table  on  opposite  page. 

—  =  negative    +  =  positive,   O  =  no  change,   A  =  acid,   Alk.  =  alkaline,  G  —  6««, 
Fl  =  fluorescence,  Pep  =  peptonization,   i=  litmus  Russell's  medium  not  reduced; 
2  =  reduced,   3  =  action   variable.     Proteus  gives  a  peculiar  dark,   heavy  oil-li1 
fluorescence  in  neutral  red  glucose  bouillon. 


Remarks 

Found  in  faeces  and  sewage-contami- 
nated water.  Differs  from  B.  typhos. 
by  marked  alkali  production. 

Blood  cultures  first  week  —  agglutina- 
tion afterward. 

Nonacid  strain,  highly  toxic. 

Acid  mannite  strain,  moderate  toxicity. 

Much  like  Flexner  strain.  No  acid  in 
maltose. 

Found  in  summer  diarrhoea  of  children. 

Little  gas.  No  fluorescence  n.  red. 
Litmus  milk  acid  in  third  day.  i. 

Much  gas.  Marked  reduction  n.  red 
with  yellow  fluorescence.  Litmus 
milk  alkaline  third  day.  2. 

B.  choleras  suis,  B.  icteroides,  B.  of 
Danysz  virus  and  B.  paratyphoid  B. 
closely  related  (Gaertner  group).  2. 

There  is  also  a  B.  coli  anaerogenes  which 
is  like  B.  coli  but  does  not  form  gas. 

Very  nearly  related  to  Friedlander's 
bacillus  as  well  as  to  B.  coli.  3. 

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gelatin  and  shows  slow  production 
of  gas  in  lactose. 

Three  types  —  Proteus  vulgaris  rapid 
gelatin  liq.;  P.  mirabilis,  slow  gelatin 
fiq.;  P.  zenkeri,  no  gelatin  liq.  Spread- 
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